JP6685900B2 - Modified hematopoietic stem / progenitor cells and non-effector T cells and uses thereof - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 61 / 898,387, filed October 31, 2013, which is incorporated by reference in its entirety.

  Hematopoietic stem / progenitor cells (HSPCs) and / or non-effector T cells (i) an extracellular component comprising a ligand binding domain, which binds a cell marker selectively expressed in unwanted cells, and (ii) an effector domain And an intracellular component containing the gene are genetically modified to express. With a variety of uses, among others, the modified cells need not be immunologically compatible prior to administration and can be administered to patients by targeting unwanted cancer cells.

  Significant advances have been made in genetically modified T cells of the immune system that target and kill unwanted cell types such as cancer cells. For example, a T cell expresses a gene having an extracellular component that binds a specific target antigen and a molecule having an intracellular component that directs the action of the T cell when the extracellular component binds to the target antigen. Have been modified. As an example, the extracellular component can be designed to bind a target antigen found on a cancer cell, and, when bound, the intracellular component can destroy the bound cancer cell. To T cells. Examples of such molecules include genetically modified T cell receptors (TCRs) and chimeric antigen receptors (CARs).

  While genetically modified T cells have significantly advanced their ability to target and destroy unwanted cell types, they can be immunologically matched to individual, specific subjects prior to use in therapeutic intervention. is necessary. Once a donor match is known (or T cells are obtained from a subject in need of treatment), the cells must be modified and expanded before use in that subject. This time consuming and costly process can cause fatal delays in treatment, for example.

  The present disclosure provides genetically modified stem cells that can be administered therapeutically without the need for immunocompatibility with a particular subject. Thus, these genetically modified stem cells provide a "ready to use" treatment that eliminates delays and costs in treatments associated with donor identification and subsequent cell modification and proliferation. The modified stem cells can be administered alone or can be used in various combinations with other therapies to achieve multiple therapeutic purposes. In certain embodiments, the modified stem cells differentiate into modified non-effector T cells prior to administration.

  More specifically, hematopoietic stem / progenitor cells (HSPCs) are extracellular components that bind specific cell markers selectively found in unwanted cell types, and when the extracellular components bind the cell markers, It is genetically modified to express a molecule having an intracellular component that directs the action of the genetically modified cell. As an example, the extracellular component can be designed to selectively bind a cell marker found on a cancer cell, and when bound, the intracellular component can bind the bound cancer cell. Instruct the genetically modified cells to destroy. Examples of such molecules include genetically modified T cell receptors (TCRs), chimeric antigen receptors (CARs) and other molecules disclosed herein. In certain embodiments, the modified HSPCs are capable of differentiating into non-effector T cells prior to administration.

Nucleotide sequence of anti-CD19 short spacer chimeric receptor, GMCSFRss-CD19scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt. Amino acid sequence of GMCSFRss-CD19scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt. Amino acid sequence of GMCSFRss-CD19scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt. Amino acid sequence of GMCSFRss-CD19scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt. Figure 3A shows a section map and amino acid sequence of ZXR-014 nucleotides. FIG. 3B shows the sequences of exemplary primers. Amino acid sequence and section map of Uniprot P0861 IgG4-Fc. Amino acid sequence and section map of Uniprot P10747 CD28. Amino acid sequence and section map of Uniprot Q070114-1BB. Uniprot P20963 human CD3ζ isoform 3 amino acid sequence and section map. An exemplary hinge arrangement. Sequence of R12 long spacer CAR: PJ_R12-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of R12 long spacer CAR: PJ_R12-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of R12 long spacer CAR: PJ_R12-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of R12 long spacer CAR: PJ_R12-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of leader_R12-hinge-CH2-CH3-CD28tm / 41BB-Z-T2A-tEGFR. Sequence of R12 intermediate spacer CAR: PJ_R12-CH3-41BB-Z-T2A-tEGFR. Sequence of R12 intermediate spacer CAR: PJ_R12-CH3-41BB-Z-T2A-tEGFR. Sequence of R12 intermediate spacer CAR: PJ_R12-CH3-41BB-Z-T2A-tEGFR. Sequence of R12 intermediate spacer CAR: PJ_R12-CH3-41BB-Z-T2A-tEGFR. Sequence of leader_R12-hinge-CH3-CD28tm / 41BB-Z-T2A-tEGFR. Sequence of R12 short spacer CAR: PJ_R12-hinge-41BB-Z-T2A-tEGFR. Sequence of R12 short spacer CAR: PJ_R12-hinge-41BB-Z-T2A-tEGFR. Sequence of R12 short spacer CAR: PJ_R12-hinge-41BB-Z-T2A-tEGFR. Sequence of R12 short spacer CAR: PJ_R12-hinge-41BB-Z-T2A-tEGFR. Sequence of leader_R12-CD28tm / 41BB-Z-T2A-tEGFR. Sequence of R11 long spacer CAR: PJ_R11-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of R11 long spacer CAR: PJ_R11-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of R11 long spacer CAR: PJ_R11-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of R11 long spacer CAR: PJ_R11-CH2-CH3-41BB-Z-T2A-tEGFR. Sequence of leader_R11-hinge-CH2-CH3-CD28tm / 41BB-Z-T2A-tEGFR. Sequence of R11 intermediate spacer CAR: PJ_R11-CH3-41BB-Z-T2A-tEGFR. Sequence of R11 intermediate spacer CAR: PJ_R11-CH3-41BB-Z-T2A-tEGFR. Sequence of R11 intermediate spacer CAR: PJ_R11-CH3-41BB-Z-T2A-tEGFR. Sequence of R11 intermediate spacer CAR: PJ_R11-CH3-41BB-Z-T2A-tEGFR. Sequence of leader_R11-hinge-CH3-CD28tm / 41BB-Z-T2A-tEGFR. Sequence of R11 short spacer CAR: PJ_R11-41BB-Z-T2A-tEGFR. Sequence of R11 short spacer CAR: PJ_R11-41BB-Z-T2A-tEGFR. Sequence of R11 short spacer CAR: PJ_R11-41BB-Z-T2A-tEGFR. Sequence of R11 short spacer CAR: PJ_R11-41BB-Z-T2A-tEGFR. Sequence of leader_R11-hinge-CD28tm / 41BB-Z-T2A-tEGFR. An exemplary spacer sequence. Sequence of Her2 short spacer construct, GMCSFss-Her2scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt. Sequence of the intermediate spacer Her2 construct. Sequence of the long spacer Her2 construct. Sequence of the long spacer Her2 construct. A library of spacer sequences. The plasmid library may include codon-optimized DNA sequences encoding extracellular components, including IgG4 hinge region, IgG4 hinge region linked to CH2 and CH3 domains, or IgG4 hinge region linked to CH3 domains. It was constructed. Any scFV sequence (VH and VL) can be cloned 5'to the sequence encoded in this diverse spacer domain library. This spacer domain is then linked to the transmembrane CD28 and intracellular signaling domain, and CD3ζ. The T2A sequence of the vector separates the chimeric receptor from a selectable marker encoding a truncated human epidermal growth factor receptor (EGFR). Design of ROR1 chimeric receptors derived from 2A2 and R12scFV with modified spacer length and different affinities (FIG. 26A). 2A2scFV, "hinge-CH2-CH3" long spacer, 229 AA, "hinge-CH3" (intermediate, 119 AA), or "hinge" only (short, 12 AA) IgG4-Fc-derived spacer, and CD3ζ and CD28. Of a lentivirus transgene insert encoding a panel of ROR1 chimeric receptors containing a signaling module having The cassette for each chimeric receptor contains a truncated EGFR marker encoded downstream of the T2A element (Figure 26B). A LOR1-specific chimeric receptor derived from R12 and 2A2 scFV with a short IgG4-Fc "hinge" spacer (12 AA), and a lentiviral transgene encoding a signal transduction module containing CD28 or 4-1BB and CD3ζ, respectively. Inserts (total of 4 constructs). Design of ROR1 chimeric receptors derived from 2A2 and R12scFV with modified spacer length and different affinities (FIG. 26A). 2A2scFV, "hinge-CH2-CH3" long spacer, 229 AA, "hinge-CH3" (intermediate, 119 AA), or "hinge" only (short, 12 AA) IgG4-Fc-derived spacer, and CD3ζ and CD28. Of a lentivirus transgene insert encoding a panel of ROR1 chimeric receptors containing a signaling module having The cassette for each chimeric receptor contains a truncated EGFR marker encoded downstream of the T2A element (Figure 26B). A LOR1-specific chimeric receptor derived from R12 and 2A2 scFV with a short IgG4-Fc "hinge" spacer (12 AA), and a lentiviral transgene encoding a signal transduction module containing CD28 or 4-1BB and CD3ζ, respectively. Inserts (total of 4 constructs). Depiction of Epitope Location on Fab of Herceptin on Epitope near Tumor Cell Membrane on Human HER2. FIG. 27B is a structural format of a spacer length variant of Herceptin scFv CAR as T2A linked to a protein having a carboxyl EGFRt marker transmembrane protein. CD-19 chimeric receptor vector. Design of lentivirus transgene inserts encoding a panel of CD-19-specific chimeric receptors that differentiate extracellular spacer lengths and intracellular stabs. In the VL-VH orientation, a CD-19-specific single chain mutant fragment derived from FMC63 mAb, hinge-CH2-CH3 (long spacer, 229 AA) or hinge alone (short spacer, 12 AA) IgG4-derived spacer domain, and Each chimeric receptor encoded by a signal transduction module containing CD3ζ or 4-1BB having CD28 in series alone or at random. Each chimeric receptor cassette contains an excised EGFR marker encoded downstream of the cleavable 2A element. Exemplary SIN lentivirus plasmid. FIG. 29A shows SIN CD19 specific scFvFc-CD3ζ CD28 CAR and huEGFRt lentiviral plasmids. Exemplary SIN lentivirus plasmid. FIG. 29B shows SIN CD19-specific scFv-4-1BBCD3ζ CAR and huEGFRt lentiviral plasmids. Percentage representation of EGFR expression as a marker of transduction efficiency / gene expression stability (FIG. 30A). HSPC were cultured on Delta as described above. On day +3, cells were introduced using the scFvFc-CD3ζCD28 CAR and huEGFRt vector with MOI of 3 in the presence of protamine sulfate, and spinfection was performed. Transgene expression was measured over the course of the culture by flow using Erbitux binding the EGFRt tag. On day 7, irradiated LCL was added to the designated cultures in a 1: 1 ratio. Absolute number display of EGFR expression as a marker of transduction efficiency / gene expression stability (FIG. 30B). HSPC were cultured on Delta as described above. On day +3, cells were introduced using the scFvFc-CD3ζCD28 CAR and huEGFRt vector with MOI of 3 in the presence of protamine sulfate, and spinfection was performed. Transgene expression was measured over the course of the culture by flow using Erbitux binding the EGFRt tag. On day 7, irradiated LCL was added to the designated cultures in a 1: 1 ratio. CD34 + CB cells cultured on Notch ligand were transduced on day 3 with a lentivirus with a MOI of 3 using the scFvFc-CD3ζ CD28 CAR and huEGFRt vector. On day 7, LCL was added to the designated medium in a 1: 1 ratio. (Transduced (■), LCL transduced (x), not transduced (cannot be widely confirmed, behind strain ■), not LCL transduced (▲)). CD34 doubling was improved by LCL addition throughout the overall TNC doubling. CD34 + CB cells cultured on Notch ligand were transduced on day 3 with a lentivirus with a MOI of 3 using the scFvFc-CD3ζ CD28 CAR and huEGFRt vector. On day 7, LCL was added to the designated medium in a 1: 1 ratio. (Transduced (■), LCL transduced (x), not transduced (cannot be widely confirmed, behind strain ■), not LCL transduced (▲)). CD34 doubling was improved by LCL addition throughout the overall TNC doubling. MIO3 on day 14 using scFv-4-1BB / CD3ζ CAR and huEGFRt vector for transduction with and without LCL. Similar growth distribution was recorded between cultures with and without LCL with LCL addition on day 7. As was done, no signs of accelerating proliferation of CAR expressing HSPC or their products appeared. The end of the culture phenotype. HSPC were cultured on Delta as described above. On day 3, designated cultures were transduced with a MOI of 3 lentivirus to express scFv-4-1BB / CD3ζ CAR and huEGFRt. Then, on day 7, designated cultures received irradiated LCL at a 1: 1 ratio. On day 14, cultures were analyzed by flow cytometry. There were no significant differences detected between transduced and non-transduced cultures. Similarly, there were no significant differences detected between the total number of cells and the EGFRt + cells suggesting that the CAR constructs were equally distributed among the subgroups. Functional analysis of scFvFc-CD3ζ CD28 CAR and huEGFRt vectors. On the final 14th day of culture on Delta, cells were removed from Delta and placed in RPMI medium supplemented with IL-2 and IL-15 for an additional week to increase NK numbers. K562 (x and ●), either propagated on Notch ligand and transduced to express the CD19-specific scFvFc-CD3ζ CD28 CAR and huEGFRt (● and ◆), or untransduced (▲ and ×), or Chromium release assay with LCL (▲ and ◆) target cells using NK effector cells derived from CD34 + CB cells. Mature NK cells were induced for an additional week in culture with RPMI, IL-2 and IL-15. Mice transplanted with transduced cells using the scFv-4-1BB / CD3ζ CAR and huEGFRt vector impaired CD19 engraftment, thus demonstrating anti-CD19 effectors and dependent on transgene expression. NOG mice transplanted with cells from a lentivirus-transduced culture encoding for scFv-4-1BB / CD3ζ CAR and huEGFRt show marked EGFRt expression and reduced CD19 engraftment.

  Significant advances have been made in genetically modified T cells of the immune system that target and kill unwanted cells, such as cancer cells. For example, a T cell expresses a gene having an extracellular component that binds a specific target antigen and a molecule having an intracellular component that directs the action of the T cell when the extracellular component binds to the target antigen. Have been modified. As an example, the extracellular component can be designed to selectively bind the target antigen found on the cancer cells, and when bound, the intracellular component can bind the bound cancer cells. Instruct T cells to destroy. Examples of such molecules include genetically modified T cell receptors (TCRs) and chimeric antigen receptors (CARs).

  Genetically modified T cells, while significantly advancing their ability to target and destroy unwanted cell types, are immunologically matched to individual, targeted subjects prior to use in therapeutic intervention. is necessary. Once a donor match is known (or T cells are obtained from a subject in need of treatment), the cells must be modified and expanded before being used in that subject. This time-consuming and expensive process can cause fatal delays in treatment, for example.

  The present disclosure provides genetically modified stem cells that can be administered therapeutically without the need for immunological adaptation to a particular subject. Thus, these genetically modified stem cells provide a "ready to use" treatment that eliminates delays and costs in treatments associated with donor identification and subsequent cell modification and proliferation. The modified stem cells can be administered alone or can be used in various combinations with other therapies to achieve multiple therapeutic purposes. In certain embodiments, the modified stem cells are capable of differentiating into modified non-effector T cells prior to administration.

  More specifically, hematopoietic stem / progenitor cells (HSPC) are characterized by the extracellular component that binds a specific cell marker and the action of the genetically modified cell when the extracellular component binds the cell marker. Are genetically modified to express a molecule having an intracellular component that directs. As an example, the extracellular component can be designed to selectively bind a cell marker found on a cancer cell, and when bound, the intracellular component can bind the bound cancer cell. Instruct the genetically modified cells to destroy. Examples of such molecules include genetically modified T cell receptors (TCRs), chimeric antigen receptors (CARs), and other molecules disclosed herein. The modified HSPC can differentiate into non-effector T cells before administration.

  As an example use of certain embodiments, cord blood transplant (CBT) is the standard of care for recurrent childhood acute lymphoblastic leukemia (ALL) when no suitable matched donor can be identified. This method is particularly important for patients with a minority or mixed ethnic background (and 30% of Caucasians) who are very unlikely to find suitable donors.

  The ability of CBT to eradicate ALL and provide long-term remission is due in part to the graft-versus-leukemia (GVL) effect. However, the recurrence rate of ALL after CBT treatment is still about 40%, including the overall survival rate associated with both relapse and treatment-related death, including graft-versus-host disease (GVHD) (Smith et al. , Biol Blood March Transplant, 2009.15 (9): p. 1086-93, Tomblyn et al., J Clin Oncol, 2009.27 (22): 3634-41). The compositions and formulations disclosed herein can enhance GVL effects without increasing the GVHD disease rate. This strategy is clinically feasible using in vitro proliferation of cord blood (CB) HSPCs via activation of the intrinsic Notch signaling pathway using Notch ligands, and more than 100-fold expansion of CD34 + cells. It gives even greater results. Clinically, this expanded HSPC can be infused with a non-engineered unit, leading to a transient engraftment of this expanded HSPC with products derived from that expanded unit, while long-term engraftment is associated with this non-manipulated unit. Finally derived from the operating unit.

  Notch ligand expanded CB HSPC is suitable for genetic modification using a vector expressing a CD19-specific CAR. This Notch ligand CB proliferation system can be utilized to genetically integrate GVL into CBT by genetically modifying proliferative HSPC to express CD19 CAR, where the transplanted myeloid and lymphoid effector cells Recognize and lyse residual leukemia cells.

  The claimed invention will now be described more generally.

  Hematopoietic stem / progenitor cells or HSPC refer to hematopoietic stem cells and / or hematopoietic progenitor cells. HSPCs can be self-renewing or (i) myeloid progenitors that ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, red blood cells, megakaryocytes / platelets or dendritic cells. The cells, or (ii) T-cells, B-cells, and lymphoid cells called natural killer cells (NK cells) can be differentiated into the finally giving rise to lymphocyte progenitor cells. For a general discussion of hematopoiesis and HSPC differentiation, see Chapter 17, Differentiated Cells and the Maintenance of Tissues, Alberts et al. , 1989, Molecular Biology of the Cell, 2nd Ed. , Garland Publishing, New York, NY, Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 5,2006, and Chapter 5 of Hematopoietic Stem Cells, 2009, Stem Cell Information, Department of Health and Human Services of the reference thing.

  HSPC can be positive for certain markers expressed at elevated levels on HSPC as compared to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or combinations thereof. Also, the HSPC can be relatively negative for expression markers as compared to other types of hematopoietic cells. For example, such markers include Lin, CD38, or a combination thereof. Desirably, the HSPC are CD34 + cells.

  Sources of HSPC include cord blood, placental blood, and peripheral blood (US Pat. Nos. 5,004,681, 7,399,633 and 7,147,626, Craddock et al., 1997, Blood 90 (12): 4779-4788, Jin et al., 2008, Journal of Translational Medicine 6:39, Pelus, 2008, Curr. Opin. Hematol. 15 (4): 285-292, Papayananopoulou. , Blood 91 (7): 2231-2239, Tricot et al., 2008, Haematologica 93 (11): 1739-1742, and Weaver et al., 2001, Bone Marro. Transplantation 27 (2): see S23-S29,). Methods such as blood sample collection, anticoagulation and treatment thereof are known to those of skill in the art. For example, Alsever et al. , 1941, N .; Y. St. J. Med. 41: 126, De Gowin, et al. , 1940, J .; Am. Med. Ass. 114: 850, Smith, et al. , 1959, J .; Thorac. Cardiovasc. Surg. 38: 573, Rous and Turner, 1916, J. Am. Exp. Med. 23: 219, and Hum, 1968, Storage of Blood, Academic Press, New York, pp. See 26-160. HSPC sources also include bone marrow (see Kodo et al., 1984, J. Clin Invest. 73: 1377-1384), germ cells, aortic / gonadal / mesonephric derived cells, lymph, liver from age-matched donors. , Thymus, and spleen. All collected samples of HSPC can be screened for unwanted components and discarded, processed, or utilized according to current accepted standards.

  HSPCs can be collected and isolated from samples using any suitable technique. Suitable collection and isolation procedures include magnetic separation, fluorescence activated cell sorting (FACS; Williams et al., 1985, J. Immunol. 135: 1004, Lu et al., 1986, Blood 68 (1): 126-133), affinity chromatography, cytotoxic drugs used in combination with monoclonal antibodies, or in combination with monoclonal antibodies such as complement and cytotoxin, "planning" with antibodies attached to a solid matrix. (Broxmeyer et al., 1984, J. Clin. Invest. 73: 939-953), selective aggregation using lectins such as soybean (Reisner et al., 1980, Proc. Natl. Acad. Sci. U.S. A.77: 1164) and the like.

  In certain embodiments, an HSPC sample (eg, a fresh cord blood unit) is loaded onto a magnetic cell separator, eg, a CliniMACS® cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). Anti-CD34 antibodies conjugated directly or indirectly can be used to select / enrich CD34 + cells. See Section 5.4.1.1 of US Pat. No. 7,399,633, which describes the enrichment of CD34 + HSPC from 1-2% to 50-80% of normal bone marrow cell numbers.

  Similarly, HSPC expressing CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof can be enriched using antibodies to those antigens. US Pat. No. 5,877,299 further describes suitable hematopoietic antigens that can be used to isolate, collect and enrich HSPC cells from a sample.

  HSPCs can be expanded to increase the number of HSPCs by the following isolation and / or enrichment. Isolation and / or growth methods are described, for example, in US Pat. Nos. 7,399,633 and 5,004,681, US Patent Publication No. 2010/01863564, International Publication (WO) WO04 / 047569, WO2007 / 0595594. , WO2011 / 127470, and WO2011 / 127472, Vumum-Finney et al. , 1993, Blood 101: 1784-1789, Delaney et al. , 2005, Blood 106: 2693-2699, Ohishi et al. 2002, J. Am. Clin. Invest. 110: 1165-1174, Delaney et al. , 2010, Nature Med. 16 (2): 232-236, and Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, and in the reference section of this specification. Each of the methods referred to in collecting, isolating, and growing is available in certain embodiments of the present disclosure.

  A preferred method of growing HSPCs is to grow HSPCs with Notch agonists. For information regarding growth of HSPC using Notch agonists, see Sections 5.1 and 5.3 of US Pat. No. 7,399,633, US Pat. Nos. 5,780,300, 5,648,464, 5,849,869, and 5,856,441, WO1992 / 119734, Schlondorfand Blobel, 1999, J. Am. Cell Sci. 112: 3603-3617, Olkkonen and Steinmark, 1997, Int. Rev. Cytol. 176: 1-85, Kopan et al. , 2009, Cell 137: 216-233, Rebay et al. , 1991, Cell 67: 687-699, and Jarriault et al. , 1998, Mol. Cell. Biol. 18: 7423-7431. In certain embodiments, the Notch agonist is immobilized during growth.

  Notch agonists include any compound that binds to or interacts with Notch proteins or other proteins in its niche pathway, thus promoting Notch pathway activation. Exemplary Notch agonists are extracellular components that bind the ligands Delta and Serrate (eg, Jagged), the hairless RBP JκI suppressor gene, Deltex, Fringe, or fragments thereof that promote activation of the Notch pathway. Nucleic acid and amino acid sequences of Delta family members and Serrate family members have been isolated from multiple species and are described in, for example, WO1993 / 12141, WO1996 / 27610, WO1997 / 01571, and Gray et al. , 1999, Am. J. Path. 154: 785-794.

In a particular embodiment, the Notch agonist is Delta1 ^ext-IgG . In certain embodiments, the Delta1 ^ext-IgG is at a concentration between 0.2 and 20 μg / ml, at a concentration between 1.25 and 10 μg / ml, or between 2 and 6 μg / ml. Concentration applied to solid phase.

  In certain embodiments, during expansion, HSPC are cultured in the presence of Notch agonist and aryl hydrocarbon receptor antagonist. The Notch agonist can be immobilized and the aryl hydrocarbon receptor antagonist can be present in the fluid in contact with the cells.

  As will be appreciated by those in the art, additional culture conditions include angiopoietin-like proteins (Angptls, eg Angptl2, Angptl3, Angptl7, Angpt15, and Mfap4), erythropoietin, fibroblast growth factor 1 (FGF-1), Flt-3 ligand (Flt-3L), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), insulin growth factor 2 (IFG-2), interleukin-3 (IL). -3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-11 (IL-11), stem cell factor (SCF, also c-kit ligand or mast cell growth factor) , Known as thrombopoietin (TPO), and (Wherein the analogs include any structural variant of a growth factor having the biological activity of a naturally occurring growth factor; see, eg, WO2007 / 1145227, US Patent Publication No. 2010/0183564). Proliferation in the presence of one or more growth factors, such as

In certain embodiments, an amount or concentration of a growth factor suitable for HSPC proliferation is an amount or concentration effective to promote proliferation of HSPC but not subsequent differentiation of that HSPC. The cell number is also preferably expanded until a suitable number of cells to give at least one infusion to a human subject is obtained, usually about 10 ⁴ cells / kg to 10 ⁹ cells / kg.

  The amount or concentration of growth factor suitable for HSPC growth depends on the activity of the growth factor preparation and the species correspondence between the growth factor and HSPC. Generally, when the growth factor and HSPC are of the same species, the total amount of growth factor in the medium is in the range of 1 ng / ml to 5 μg / ml and in the range of 5 ng / ml to 1 μg / ml. , Or in the range of 5 ng / ml to 250 ng / ml. In a further embodiment, the amount of growth factor may be in the range 5-1000 or 50-100 ng / ml.

  In certain embodiments, the aforementioned growth factors are present in the culture conditions for HSPC growth at the following concentrations. 25-300 ng / ml SCF, 25-300 ng / ml Flt-3L, 25-100 ng / ml TPO, 25-100 ng / ml IL-6 and 10 ng / ml IL-3. In a more particular embodiment, 50, 100 or 200 ng / ml SCF, 50, 100 or 200 ng / ml Flt-3L, 50 or 100 ng / ml TPO, 50 or 100 ng / ml IL. -6, and 10 ng / ml IL-3 can be used.

  In certain embodiments, HSPC comprises one fixed Notch agonist and 50 ng / ml or 100 ng / ml SCF, one fixed Notch agonist, and 50 ng / ml or 100 ng / ml. Flt-3L, IL-6, TPO, and SCF each, or one fixed Notch agonist and 50 ng / ml or 100 ng / ml Flt-3L, IL-6, TPO, and SCF each, It can then be propagated by exposing the HSPC to 10 ng / ml IL-11 or IL-3.

  HSPC is fibronectin (FN), or a fragment thereof (for example, CH-296 (Dao et. Al., 1998, Blood 92 (12): 4612-21)) or RetroNectin (registered trademark) (gene recombinant human fibronectin. It can be grown in tissue culture dishes of cultures to which extracellular matrix proteins have been bound, such as fragments (Clontech Laboratories, Inc., Madison, WI).

In certain embodiments, the HSPC expansion method comprises immobilized Delta1 ^ext-IgG and CH-296 and four or more growth factors selected from IL-6, TPO, Flt-3L, CSF, and IL-3. Methods include culturing exogenously isolated HSPC on a coated solid phase, thus producing a expanded HSPC sample.

  In certain embodiments of growing HSPC, the cells are loaded with immobilized Delta ligand and fibronectin and 25 ng / ml or 100 ng / ml (or any range between these values), and preferably 50 ng / ml. It is cultured on a plastic tissue culture dish containing each of SCF and TPO. In a particular embodiment of growing HSPC, the cells are 25 ng / ml or 100 ng / ml (or any range between these values), and preferably 50 ng / ml of SCF and Flt-3L, respectively. Cultured on a plastic tissue culture dish containing immobilized Delta ligand and fibronectin in the presence of In certain embodiments of growing HSPC, the cells are loaded with immobilized Delta ligand and fibronectin and 25 ng / ml or 100 ng / ml (or any range between these values), and preferably 50 ng / ml. It is cultured on a plastic tissue culture dish containing each of SCF, Flt-3L and TPO. In certain embodiments of growing HSPC, the cells are loaded with immobilized Delta ligand and fibronectin and 25 ng / ml or 100 ng / ml (or any range between these values), and preferably 50 ng / ml. It is cultured on a plastic tissue culture dish containing each of SCF, Flt-3L, TPO, and IL-6. In certain embodiments, the HSPCs are further cultured in the presence of 5 to 15 ng / ml, and preferably 10 ng / ml IL-3. In a particular embodiment, the HSPCs are further cultured in the presence of 5 to 15 ng / ml, and preferably 10 ng / ml GM-CSF. In certain embodiments, the one or more growth factors used are not GM-SCF or IL-7. In certain compatible embodiments, fibronectin is omitted from the tissue culture dish or replaced with another extracellular matrix protein. Further methods and details regarding the growth of HSPC can be found in WO 2013/086436.

  In certain embodiments, the percentage of CD34 + cells in expanded HSPC samples obtained using the described method is higher than the percentage of CD34 + cells in isolated HSPC before expansion. For more information on suitable culture conditions, see US Pat. No. 7,399,633, US Patent Publication No. 2010/0183564, and Freshney Culture of Animal Cells, Wiley-Liss, Inc. , New York, NY (1994)).

  Modified HSPC. In certain embodiments, HSPCs are modified to express a molecule that has one extracellular component and one intracellular component. The extracellular and intracellular components can be linked to one transmembrane domain, one tag sequence, and / or one linker sequence, either directly or via a spacer region.

  Extracellular component. The extracellular component comprises at least one ligand linking the domains (hereinafter the binding domain). The binding domain is designed such that the modified cell targets a particularly unwanted cell type by binding a cellular marker that is selectively found on the unwanted cell type.

  Cell marker. In certain embodiments, cell markers are selectively expressed by unwanted cells, such as unwanted cancer cells. "Selectively expressed" means that one cell marker is found at higher levels on one unwanted cell type as compared to other non-target cells. Within sound medical judgment, the administration of cells that target and kill the unwanted cells based on the presence of the marker entrains another non-target cell that may express the marker to a lesser degree. The difference in such expression levels, which outweighs the risk of causing it, is very important. In certain instances, cell markers are expressed only by unwanted cell types. In other examples, the cell marker is on at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95% on the undesired cell type as compared to non-target cells. It is highly expressed by 96%, 97%, 98%, 99%, or 100% or more. Exemplary undesirable cancer cells include adrenal gland cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, epithelial malignancy, cervical cancer, colon cancer, colon cancer, corpus. Uterine cancer, ear, nose and throat (ENT) cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, Hodgkin's disease, bowel cancer, kidney cancer, larynx Cancer, leukemia, liver cancer, lymph node cancer, lymphoma, lung cancer, melanoma, mesothelioma, myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, ovarian cancer , Pancreatic cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, non-epithelial malignant tumor, seminoma, skin cancer, gastric cancer, teratoma, testicular cancer, thyroid cancer, uterus , Vaginal cancer, vascular tumors, and cancer cells from their metastases.

Certain cancer cells below can be targeted by including within their extracellular components a domain that binds the relevant cellular markers.

  Without being limited to the above, cell markers also include A33, BAGE, Bcl-2, β-catenin, B7H4, BTLA, CA125, CA19-9, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD44v6, CD45, CD123, CEA, CEACAM6, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ephrin B2, ErbB2, ErbB3, ErbB4, EphA2, estrogen receptor, FAP, ferritin, α-fetoprotein (AFP). , FLT1, FLT4, folate binding protein, Frizzled, GAGE, G250, GD-2, GHRHR, GHR, GM2, gp75, gp100 (Pmel 17), gp130, HLA, HER-2 / neu, HPV E6, PV E7, human telomerase reverse transcriptase, HVEM, IGF1R, IL6R, KDR, Ki-67, LIFRβ, LRP, LRP5, LTβR, mesothelin, OSMRβ, p53, PD1, PD-L1, PD-L2, PRAME, progesterone receptor , PSA, PSMA, PTCH1, MAGE, MART, mesothelin, MUC, MUC1, MUM-1-B, myc, NYESO-1, RANK, ras, Robo1, RORl, survivin, TCRα, TCRβ, tenascin, TGFBR1, TGFBR2, TLR. , TLR9, TNFR1, TNFR2, TNFRSF4, TWEAK-R, TSTA tyrosinase, VEGF, and WT1.

Cell markers for specific cancer cells include:

  Undesired cells and cell markers are not limited to cancer cells and cancer cell markers, but can also include virus-infected cells, such as those expressing the hepatitis B surface antigen.

  Binding domain. The binding domain includes any substance that binds a cell marker that forms a complex. Examples of binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (eg T cell receptors), or combinations thereof.

  Antibodies are an example of a binding domain and all antibodies or antibody-binding fragments, such as Fv, Fab, Fab ', F (ab') 2, Fc, and single chain (sc) formers and cells. Includes those fragments that specifically bind the marker. Further exemplifications include scFv-based grabbers and soluble VH domain antibodies. These antibodies form a binding region that uses only the variable region of the heavy chain. For example, Jespers et al. , Nat. Biotechnol. 22: 1161, 2004, Cortez-Retamozo et al. , Cancer Res. 64: 2853, 2004, Baral et al. , Nature Med. 12: 580, 2006, and Barthelemy et al. J. Biol. Chem. 283: 3639, 2008).

  Antigens binding antibodies or fragments include all or part of polyclonal, monoclonal, human, humanized, synthetic, chimeric, bispecific, mini, and linear antibodies. be able to.

  Antibodies of human origin or humanized antibodies have low or no immunogenicity in humans and have a low number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and fragments thereof are generally selected to have low or no levels of antigen in human subjects.

  Antibodies that specifically bind a particular cell marker can be recombinant to produce monoclonal antibodies, phage display methods, human or humanized antibody production methods, or antibodies known to those of skill in the art. Methods using transgenic animals or plants (see, eg, US Pat. Nos. 6,291,161 and 6,291,158). Libraries for phage display of some or all synthetic antibodies are available and antibodies or fragments thereof capable of binding cell markers can be selected. For example, the binding domain may be identified by selecting a Fab phage library that corresponds to the Fab fragment that specifically binds the cellular marker of interest (Hoet et al., Nat. Biotechnol. 23: 344, 2005). Libraries for phage display of human antibodies are also available. Furthermore, a simple strain (for example, mouse, HuMAb mouse (registered trademark) (GenPharm Int ″ l. Inc., Mountain View, CA) TC mouse (registered trademark) (Kirin Pharma Co. Ltd., Tokyo, JP), KM-mouse (R) (Medarex, Inc., Princeton, NJ) Lama, chicken, rat, hamster, rabbit, etc.) has a traditional strategy corresponding to the expression of hybridoma using a cell marker of interest as an immunogen. , Can be used to express the binding domain. In certain embodiments, the antibody binds a cellular marker that is selectively expressed by certain unwanted cell types and does not cross-react with non-specific components or unrelated targets. Once identified, the amino acid sequence of the antibody and the gene sequence encoding the antibody can be isolated and / or determined.

  Alternative sources of binding domains include scTCRs (eg, Lake et al., Int. Immunol. 11: 745, 1999, Maynard et al., J. Immunol. Methods 306: 51, 2005, US Pat. 794), a fibrinogen domain (see, eg, Weisel et al., Science 230: 1388, 1985), a Kunitz domain (see, eg, US Pat. No. 6,423,498), a designed ankyrin repeat protein. (DARPins; Binz et al., J. Mol. Biol. 332: 489, 2003, and Binz et al., Nat. Biotechnol. 22: 575, 2004), fibronectin binding domain (Adne. Chin or monobodies, Richards et al., J. Mol. Biol. 326: 1475, 2003, Parker et al., Protein Eng. Des. Selec. 18: 435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381: 1238-1252), cysteine-knot miniprotein (Vita et al., 1995, Proc. Nat'l. Acad. Sci. (USA) 92: 6404-6408; Martin et al., 2002, Nat. Biotechnol. 21:71, 2002, and Huang et al. (2005) Structure 13: 755, 2005), the repeating domain of the tetratricopeptide (Main et al. , Structure 11: 497, 2003 and Cortajarena et al., ACS Chem. Biol. 3: 161, 2008, leucine-rich repeat domain (Stumpp et al., J. Mol. Biol. 332: 471, 2003), lipocalin. Domains (e.g. WO 2006/095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96: 1898, 1999, and Schonfeld et al., Proc. Nat'l. Acad. Sci. USA 106: 8198, 2009), V-like domains (see, e.g., U.S. Patent Publication No. 2007/0065431), C-type lectin domains (Zelensky and Gready, FEBS). . 272: 6179, 2005, Beavil et al. , Proc. Nat'l. Acad. Sci. (USA) 89: 753, 1992 and Sato et al. , Proc. Nat'l. Acad. Sci. (USA) 100: 7779, 2003), mAb2 or Fcab brand (see, for example, WO 2007/098934 and WO 2006/072620), armadillo repeat proteins (eg, Madhurantanakam et al., Protein Sci. 21: 1015, 2012). , WO 2009/040338), Affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), Affibody, Avimers, Knottins, Finomers, fynomers. Attrimers, cytotoxic T lymphocyte-related protein-4 (Weidle et al., Cancer Gen. Proteo. 10: 155, 2013), or the like (Nord et al., Protein Eng. 8: 601, 1995, Nord et al., Nat. Biotechnol. 15: 772, 1997, Nord et al., Euro. J. Biochem. 268: 4269, 2001, Binz et al., Nat. Biotechnol. 23: 1257, 2005, Boersma and Pluckthun, Curr. Opin. Biotechnol. 22: 849, 2011). Sequence or a sequence encoding the recombinant diversity of amino acids in the loop region of a compatible non-antibody scaffold.

  In certain embodiments, the binding domain comprises Vα / β and Cα / β chains (eg, Vα-Cα, Vβ-Cβ, Vα-Vβ), or is linked to a cellular marker of interest (eg, peptide-MHC complex). It is a single-chain T cell receptor (scTCR) containing Vα-Cα, Vβ-Cβ, and Vα-Vβ pairs that are specific to the above.

  Peptide aptamers contain a peptide loop (specific for a cell marker) connected to both ends of the protein scaffold. This double structural constraint increases the binding affinity of peptide aptamers to levels comparable to antibodies. The variable loop length is usually 8 to 20 amino acids and the scaffold can be any, stable, soluble, small and non-toxic protein. Selection of peptide aptamers can be performed utilizing different systems such as the yeast 2-hybrid system (eg, Gal4 yeast-2-hybrid system), or the LexA interaction trap system.

In a particular embodiment, the binding domain may be an antibody that binds the cell marker CD19. In certain embodiments, the binding domain is a single chain Fv fragment (scFv) that comprises the VH and VL regions specific for CD19. In certain embodiments, the VH and VL regions are human. Exemplary VH and VL regions include a segment of anti-CD19 specific monoclonal antibody FMC63. In a particular embodiment, the scFV is a human or humanized fragment and is the sequence of CDRL1 that is RASQDISKYLN (SEQ ID NO.108), the sequence of CDRL2 that is SRLHSGV (SEQ ID NO.111), and GNTLPYTFG. It contains a light chain of the variable region, which comprises a sequence of certain CDRL3 (SEQ ID NO. 104). In another embodiment, the scFV is the sequence of CDRH1 that is DYGVS (SEQ ID NO. 103), the sequence of CDRH2 that is V I WGSETTYNYSALKS (SEQ ID NO. 114), and the sequence of CDRH3 that is YAMDYWG (SEQ ID NO. No. 115), which is a human or humanized ScFv containing a heavy chain of a variable region.

  The gene sequence encoding the binding domain is shown in Figure 1 as scFV from an antibody that specifically binds CD19, such as FMC63. The gene sequence encoding the flexible linker containing the amino acid of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 30) separates VH and VL of the scFV. The amino acid sequence of the scFv containing the linker is shown in FIG. 2 (SEQ ID NO: 34). Other CD19-targeted antibodies are known, such as SJ25C1 (Bejsek et al. Cancer Res 2005, PMID 7538901) and HD37 (Pezutto et al. JI 1987, PMID 2437199). SEQ ID NO. 10 gives the DNA sequence of anti-CD19 scFv (VH-VL) and SEQ ID NO. 9 gives the amino acid sequence of anti-CD19 scFv (VH-VL).

  In certain embodiments, the binding domain binds the cell marker ROR1. In certain embodiments, the scFV is the sequence of CDRL1 that is ASGFDFSAYYM (SEQ ID NO. 101), the sequence of CDRL2 that is TIYPSSG (SEQ ID NO. 112), and the sequence of CDRL3 that is ADRATYFCA (SEQ ID NO. 101). 100) is a human or humanized scFv comprising a variable light chain. In certain embodiments, the scFV is a sequence of CDRH1 that is DTIDWY (SEQ ID NO.102), a sequence of CDRH2 that is VQSDGSYTKRPPGVPDR (SEQ ID NO.113), and a sequence of CDRH3 that is YIGGYVFG (SEQ ID NO. 117) is a human or humanized scFv comprising the variable region heavy chain comprising 117).

  In certain embodiments, the binding domain binds the cell marker ROR1. In certain embodiments, the scFV is a sequence of CDRL1 that is SGSDINDYPIS (SEQ ID NO.109), a sequence of CDRL2 that is INSGGST (SEQ ID NO.105), and a sequence of CDRL3 that is YFCARGYS (SEQ ID NO.109). 116) is a human or humanized scFv comprising a variable light chain. In a specific embodiment, the scFV is the sequence of CDRH1 that is SNLAW (SEQ ID NO. 110), the sequence of CDRH2 that is RASNLASGVPSRFSGS (SEQ ID NO. 107), and the sequence of CDRH3 that is NVSYRTSF (SEQ ID NO. 106) is a human or humanized ScFv comprising the heavy chain of the variable region including 106). Many more antibodies specific for ROR1 are known to those of skill in the art.

  In certain embodiments, the binding domain binds the cell marker Her2. Many antibodies specific for Her2 are known to those of skill in the art and can be readily characterized in terms of sequence, epitope binding, and affinity. In certain embodiments, the binding domain comprises a scFv sequence from a Herceptin antibody. In certain embodiments, the binding domain is a human or humanized ScFv that comprises the light chain of the variable region comprising the CDRLl, CDRL2, and CDRL3 sequences of a Herceptin antibody. In certain embodiments, the scFV is a human or humanized ScFv that comprises a variable region heavy chain comprising CDRH1, CDRH2, and CDRH3 sequences of a Herceptin antibody. The CDR sequence can be easily determined from the amino acid sequence of Herceptin. Exemplary gene sequences encoding the Her2 ligand binding domain are found in SEQ ID NOs: 39 and 40.

  In certain embodiments, the CDR regions are found within the antibody region, which is counted by Kabat as follows. For the light chain, CDRL1 is amino acids 24-34, CDRL2 is amino acids 50-56, CDRL3 is amino acids 89-97, and for the heavy chain CDRH1 is amino acids 31-35, CDRH2 is amino acids. 50-65 and CDRH3 are amino acids 95-102.

  Other antibodies are known and are commercially available. For example, anti-PSMA and anti-PSCA antibodies are available from Abcam plc (ab66912 and ab15168, respectively). Mesothelin and WT1 antibodies are available from Santa Cruz Biotechnology, Inc. Anti-CD20 antibodies such as rituximab (tradenames Rituxan, MabThera and Zytux) have been developed by IDEC Pharmaceuticals.

  Intracellular components. The intracellular component that expresses the molecule can include an effector domain. Effector domains can send functional signals to cells. In certain embodiments, the effector domain enhances a cellular response, either directly or indirectly, with one or more other proteins that directly enhance the cellular response. The effector domain is capable of conferring activation of at least one function of the altered cell, which binds a cellular marker expressed on the unwanted cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or another effector function.

  Effector domains can include one, two, three or more receptor signaling domains, intracellular signaling domains (eg, cytoplasmic signaling sequences), stimulatory domains, or combinations thereof. . Exemplary effector domains include 4-1BB, CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, LAG3, LAT, Lck, It comprises a signaling and stimulatory domain selected from LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, or any combination thereof.

  Primary cytoplasmic signaling sequences that act upon stimulation may include receptor tyrosine activation motifs or signaling motifs known as iTAMs. Examples of iTAMs containing primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, and FeRγ. In certain embodiments, the variant of CD3ζ carries at least one, two, three, or all ITAM regions shown in FIG.

  In certain embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or a signal transduction domain thereof, ITAM, a majority of stimulatory domains. , Or any combination thereof.

  Examples of intracellular signaling domains include the cytoplasmic sequences of the CD3 ζ chain, and / or co-receptors that act simultaneously with the initiation of signal transduction following the involvement of the binding domain.

  In certain embodiments, the intracellular signaling domain of a molecule expressed by a modified cell can be designed to include the intracellular signaling domain in combination with any other desired cytoplasmic domain. For example, the intracellular signaling domain of a molecule can include intracellular signaling domains and costimulatory domains such as costimulatory regions.

  This costimulatory signaling region refers to the part of the molecule that contains the intracellular domain of the costimulatory domain. A costimulatory domain is a cell surface molecule other than an expressed cell marker binding domain that allows lymphocytes to respond to the binding of cell markers. Examples of such molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, lymphocyte functional-related antibody-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7. -H3, and ligands that specifically bind to CD83.

  In a particular embodiment, the amino acid sequence of the intracellular signaling domain comprising a variant of CD3ζ and a portion of the 4-1BB intracellular signaling domain is provided in FIG. Representative gene sequences are given in Figure 1 (SEQ ID NO: 16, SEQ ID NO: 1).

  In certain embodiments, the intracellular signaling domain comprises (i) all or part of the CD3ζ signaling domain, (ii) all or part of the CD28 signaling domain, and (iii) 4-1BB It comprises all or part of the signaling domain or (iv) all or part of the signaling domain of CD3ζ, CD28 and / or 4-1BB.

  The intracellular signaling domain sequences of the expressed molecule may be linked to each other randomly or in a particular order. Optionally, short oligo- or protein linkers, preferably 2 to 10 amino acids in length, may form the linkage.

  Spacer area. In a particular embodiment, the spacer region is found between its binding domain and the intracellular components of the expressed molecule. In certain embodiments, the spacer region is part of the extracellular component of the expressed molecule.

  The length of the spacer region can be customized to accommodate individual cell markers on unwanted cells to optimize recognition and destruction of unwanted cells. In certain embodiments, the length of the spacer region is a molecule that grows in vitro and / or in vivo in response to the location of a cell marker epitope, the affinity of the binding domain for that epitope, and / or the recognition of a cell marker. Can be selected according to the ability of the modified cells expressing

  Usually, the spacer region is found between the binding domain and the transmembrane domain of the expressed molecule. The spacer region may confer flexibility on its binding domain and confer high expression levels on the modified cells. In certain embodiments, the spacer region is at least 10 to 250 amino acids, at least 10 to 200 amino acids, at least 10 to 150 amino acids, at least 10 to 100 amino acids, at least 10 to 50 amino acids, or at least 10 to 25 amino acids. Can have amino acids of. In further embodiments, the spacer region is 250 or less amino acids, 200 or less amino acids, 150 or less amino acids, 100 or less amino acids, 50 or less amino acids, 40 or less amino acids, 30 or less amino acids, 20 or less amino acids, or It has 10 or less amino acids.

  In certain embodiments, the spacer region may be derived from all or part of the hinge region of an immunoglobulin-like molecule, eg, human IgG1, IgG2, IgG3, or IgG4. The hinge region can be modified to avoid unwanted structural interactions such as dimerization. In certain embodiments, all or a portion of the hinge region may be combined with one or more domains of immunoglobulin constant regions. For example, part of the hinge region can be combined with all or part of the CH2 or CH3 domain. In a particular embodiment, the spacer region does not comprise a 47-48 amino acid hinge region sequence from CD8α.

  In certain embodiments, the spacer region is in combination with all or part of the CH2 region, all or part of the CH3 region, or all or part of the CH2 region and all or part of the CH3 region. Selected from the group comprising hinge region sequences from IgG1, IgG2, lgG3, or lgG4 in.

  In certain embodiments, the short spacer region has 12 amino acids or less and comprises all or part of an IgG4 hinge region sequence (eg, a protein encoded by SEQ ID NO: 50), and the intermediate spacer region is 119 or less, and includes the IgG4 hinge region sequence and all or part of the CH3 region (eg, SEQ ID NO: 52), the long spacer region has 229 or less amino acids and the IgG4 hinge region. It includes all or part of the sequence, CH2 region, and CH3 region (eg, SEQ ID NO: 50).

  In certain embodiments, a long spacer region (eg, 229 amino acids or less and 119 amino acids or more) is selected when the binding domain binds a portion of a cellular marker that is very proximal to the membrane of the unwanted cell. Be done. Very close to the undesired cell membrane means within the first 100 extracellular amino acids of the cell marker.

  In certain embodiments, where the binding domain binds a portion of a cellular marker that is distal to the membrane of an unwanted cell, an intermediate or short pacer region is chosen (eg, 119 or less amino acids or 12 or less amino acids. ).

  As will be understood by those skilled in the art, whether the binding portion of the cell marker is proximal or distal to the membrane can be determined based on a three-dimensional structural model or crystal structure analysis.

  In certain embodiments, the expressed molecule comprises a binding domain comprising a scFV that binds to a ROR1 epitope located distal to the membrane relative to an Ig / Frizzled domain and a spacer of 15 amino acids or less. In certain embodiments, the expressed molecule comprises a binding domain comprising an scFV that binds the ROR1 epitope located proximal to the membrane with respect to the kringle domain and a spacer longer than 15 amino acids. In certain embodiments, the expressed molecule comprises a binding domain that comprises an scFV that binds CD19 and a spacer that is 15 amino acids or less.

In certain embodiments, the binding domain is (i) a CDRL1 sequence that is RASQDISKYLN (SEQ ID NO: 108), a CDRL2 sequence that is SRLHSGV (SEQ ID NO: 111), and a CDRL3 sequence that is GNTLPYTFG (SEQ ID NO). : 104) and a CDRH1 sequence (SEQ ID NO: 103) which is DYGVS, a CDRH2 sequence (SEQ ID NO: 114) which is V I WGSETTYNYSALKS, and a CDRH3 sequence (SEQ ID NO: 115) which is YAMDYWG. ), Or (ii) a CDRL1 sequence that is ASGFDFSAYYM (SEQ ID NO: 101), a CDRL2 sequence that is TIYPSSG (SEQ ID NO: 112), and ADRATYF A mutant light chain containing a CDRL3 sequence (SEQ ID NO: 100) that is A, and a CDRH1 sequence (SEQ ID NO: 102) that is DTIDWY, a CDRH2 sequence (SEQ ID NO: 113) that is VQSDGSYTKRPPGVPDR, and a CDRH3 that is YIGGYVFG. When including a mutated heavy chain comprising the sequence (SEQ ID NO: 117), the spacer may be 12 amino acids or less, and in more particular embodiments SEQ ID NO: 47.

  In certain embodiments, the binding domain is (i) a CDRL1 sequence that is SGSDINDYPIS (SEQ ID NO: 109), a CDRL2 sequence that is INSGGST (SEQ ID NO: 105), and a CDRL3 sequence that is YFCARGYS (SEQ ID NO). : 116) and a CDRH1 sequence (SEQ ID NO: 110) that is an SNLAW, a CDRH2 sequence (SEQ ID NO: 107) that is RASNLASGVPSRFSGS, and a CDRH3 sequence (SEQ ID NO: 106) that is NVSYRTSF. Or (ii) a mutant light chain comprising a Herceptin antibody CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence, and a Herceptin antibody CDRH1 sequence, a CDRH2 sequence and a CDR When comprising a variant heavy chain comprising the 3 sequences, the spacer 229 can be a less amino acids, In a more specific embodiment, SEQ ID NO: can contain 61.

  Transmembrane domain. The expressed molecules disclosed herein can also include a transmembrane domain, at least a portion of which is located between the extracellular and intracellular components of interest. This transmembrane domain can fix the expressed molecule on the membrane of the modified cell. The transmembrane domain can be derived from either natural and / or synthetic sources. When the source is natural, the transmembrane domain can be derived from any membrane bound or transmembrane protein. At least in the transmembrane domain, α, β or ζ chain of T cell receptor, CD28, CD3, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and The transmembrane region of CD154 can be included. The transmembrane domain can include those shown in FIG. 2 or FIG.

  In certain embodiments, the transmembrane domain comprises the amino acid sequence of the CD28 transmembrane domain or the CD4 transmembrane domain shown in FIG. A representative gene sequence encoding the CD28 transmembrane domain is shown in Figure 1 (SEQ ID NO: 12). SEQ ID NO: 118 is a representative gene sequence encoding the CD4 transmembrane domain.

  Tag array. In certain embodiments, the expressed molecule further comprises a tag sequence. Tag sequences can be provided for identification and / or selection of transduced cells. Many different tag sequences can be employed. The positively selective tag sequence is encoded by the gene and is introduced into the modified cell of interest to express the predominant trait limiting positive selection of cells carrying the gene. This type of gene is known to those skilled in the art, and the hygromycin B phosphotransferase gene (hph), which confers resistance to hygromycin B, the amino acid sequence from Tn5 which encodes resistance to antibiotic 0418. It includes a glycosyl transferase gene (neo or aph), a dihydrofolate reductase (DHFR) gene, an adenosine deaminase gene (ADA), and a multidrug resistance (MDR) gene. In a particular embodiment, the tag sequence is the truncated EGFR shown in FIG. An exemplary gene sequence encoding this truncated EGFR is shown in Figure 1 (SEQ ID NO: 9).

  In certain embodiments, the functional gene may be introduced into a modified HSPC that allows for negative selection in vivo. By "negative selection" is meant that the administered cells can be eliminated as a result of changes in the in vivo conditions of a subject. This negative selective trait can result from the insertion of a gene conferring sensitivity to the administered drug. Negative selective genes are known to those skilled in the art, and herpes simplex virus type I thymidine kinase (HSV-ITK) gene, cellular hypoxanthine-phosphoribosyl transferase (HPRT) gene, which confers ganciclovir sensitivity, cellular The adenine-phosphoribosyl transferase (APRT) gene, and bacterial cytosine deaminase. Further disclosures supporting negative selection include Lupton S. et al. D. et. al. , Mol. and Cell Biol. , 11: 6 (1991), Riddell et al. , Human Gene Therapy 3: 319-338 (1992), WO 1992/008796 and WO 1994/028143 and US Pat. No. 6,040,177, paragraphs 14-17).

  The design of the specific molecule expressed by the modified cell depends on the type of the target cell marker, the affinity of the binding domain for the cell marker, the flexibility required for the cell marker binding domain, and / or the intracellular signaling domain. Can be customized. In certain embodiments, numerous constructs have been tested in in vitro and in vitro models to determine the ability of the modified cells to grow in culture and / or kill unwanted cells. In certain embodiments, at least 30% of the modified effector (eg, differentiated modified HSPC) is modified so that one molecule grows in vivo for at least two generations and / or within 72 hours of introduction into the body. Are selected to provide the capabilities of. In a particular embodiment, one molecule is not selected such that greater than 50% of the cells undergo activation that induces cell death (AICD) within 72 hours in vivo in immunodeficient mice, and It is not selected to fail to reduce the presence of tumor cells.

  The following disclosure provides more specific illustrations of expressed molecules and related vectors.

  “Chimeric antigen receptor” or “CAR” refers to a synthetically engineered receptor that comprises a bound domain that binds a cellular marker that is specifically associated with unwanted cells linked to an effector domain. The binding domain and effector domain can be linked via a spacer domain, a transmembrane domain, a tag sequence, and / or a linker sequence.

  In certain embodiments, ROR1-specific and CD19-specific CARs can be constructed using the VL and VH chain segments of 2A2, R12, and R11 mAhs (ROR1) and FMC63 mAb (CD19). The sequences of the mutated regions corresponding to R11 and R12 are provided in Yang et al, Plos One 6 (6): e21018, June 15, 2011. Each scFV depends on the (G4S) 3 (SEQ ID NO: 60) protein, resulting in "hinge-CH2-CH3" (229 AA, SEQ ID NO: 61) and "hinge-CH3" (119 AA, SEQ ID NO: 52). ) Or a “hinge alone” (12 AA, SEQ ID NO: 47) sequence (FIG. 1), in a spacer domain derived from IgG4-Fc (Uniprot database: P01861, SEQ ID NO: 92). Be connected. All spacers can include an S → P substitution within the “hinge” domain located at position 108 of the native IgG4-Fc protein, and 27AA of human CD28 (Uniprot: P10747, SEQ ID NO: 93). 41AA cytoplasmic domain of human CD28 with a LL → GG substitution located in the transmembrane domain and (i) at positions 186-187 of the native CD28 protein (SEQ ID NO: 93), or (ii) human 4-1BB (Uniprot). : Q07011, SEQ ID NO: 95), each of which may be linked to a signaling module of an effector domain, each of which is an isoform of human CD3ζ (Uniprot: P20963, SEQ ID NO: 94). Form 3 112AA thin It is connected to the quality domain. This construct encodes the skip element of the T2A ribosome (SEQ ID NO: 88) and the tEGFR sequence downstream of the chimeric receptor (SEQ ID NO: 27). Codon-optimized gene sequences encoding each transgene can be synthesized (Life Technologies) and cloned into an epHIV7 lentiviral vector using Nhel and Not1 restriction sites. This epHIV7 lentiviral vector can be derived from the pHIV7 vector by replacing the cytomegalovirus promoter pHIV7 with the EF-1 promoter. The tEGFR encoding ROR1 chimeric receptor, CD19 chimeric receptor or lentivirus is expressed in packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G, and 293T cells using Calphos® transfection reagent (Clontech). Can be produced in

  A HER2-specific chimeric receptor can be constructed using VL and VH chain segments of a HER2-specific mAb that recognizes a membrane-proximal epitope on HER2 (FIG. 12A), and its scFVs are IgG4 hinge / CH2 / CH3, It can be linked to the extracellular spacer domain of IgG4 hinge / CH3, and IgG4 hinge alone, and to the CD28 transmembrane domain, 4-1BB and CD3ζ signaling domain (FIG. 12B).

  As shown, each CD19 chimeric receptor has a sequence of CD19-specific mAb FMC63 (scFv: VL-VH), a'hinge-CH2-CH3 'domain (229 AA, long spacer) or a'hinge', either alone or in parallel with each other. Corresponding to an IgG4-Fc-derived spacer containing either domain alone (12 AA, short spacer), and a CD3ζ signaling module with membrane-proximal CD28 or 4-1BB costimulatory domains (FIG. 13A). , Single-chain mutant fragments. The transgene cassette is separated by a cleavable T2A element and cleaved from the chimeric receptor gene to serve as a tag sequence for transduction, selection and in vivo tracking of the chimeric receptor modified cells. tEGFR) downstream.

  As will be appreciated by those in the art, the modified HSPC can be recombined by introducing a recombinant gene sequence into the HSPC. A description of genetically modified HSPC can be found in Section 5.1 of US Pat. No. 7,399,633. A gene that is desirably expressed in the modified cell is introduced into the HSPC that is expressible by the cell and / or their products.

  The desired gene is transfection, electroporation, microinjection, lipofection, calcium phosphate mediated gene transfer, infection with a viral or bacteriophage vector containing the gene sequence, cell fusion, chromosomal mediated gene transfer, microcell mediated It can be introduced into HSPC by any method known in the art, such as gene transfer, spheroplast fusion and the like. Numerous techniques for introducing foreign genes into cells are known in the art (eg, Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-618, Cohen et al., 1993, Meth. Enzymol. 217: 618-644, Cline, 1985, Pharmac. Ther. 29: 69-92) and the expression and physiological functions required for recipient cells are utilized without being disrupted. In order for the gene to be expressed by the cell and more preferably inherited and expressed by the product of the cell, techniques for properly introducing the gene into the cell are essential. As indicated, in certain embodiments, the introducing method comprises introducing a selectable tag sequence into the cell. The cells are then sorted to accept the transfection and to isolate cells expressing the transgene.

  The term "gene" refers to a nucleic acid sequence (used interchangeably with a polynucleotide or nucleotide sequence) that encodes a molecule having extracellular and intracellular components described herein. This definition includes polymorphisms, mutations, and / or such alterations of the various sequences that do not substantially adversely affect the function of the encoded molecule. The term “gene” may include coding sequences as well as constant regions such as promoters, enhancers and terminal regions. The term can further include all introns and other DNA sequences spliced from the mRNA transcript, with variants resulting from compatible splice sites. The gene sequence encoding the molecule can be DNA or RNA that directs expression of the molecule. These nucleic acid sequences may be DNA standard sequences transcribed into RNA or RNA sequences translated into proteins. Such nucleic acid sequences include both full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequence can also include degenerate codons, either the native sequence or a sequence introduced to confer codon preferences for a particular cell type. It will be appreciated by those skilled in the art that a portion of the complete gene sequence will be referenced throughout this disclosure.

  A gene sequence encoding a binding domain, effector domain, spacer region, transmembrane domain, tag sequence, linker sequence, or any other protein or peptide sequence described herein is synthesized or assembled from related amino acid sequences. It can be easily prepared by the replacement method. In some embodiments, the gene sequences encoding any of these sequences also have their coding sequences to facilitate excision and replacement of gene sequences encoding sequences with other gene sequences encoding different sequences. It can have one or more restriction enzyme sites at the 5'and / or 3'ends of. In embodiments, the gene sequence encoding the sequence may be codon optimized for expression in mammalian cells.

  “Encode” refers to the characteristic of a specific sequence of nucleotides within a gene, such as cDNA or mRNA, and serves as a template for the synthesis of other macromolecules, such as sequences defined as amino acids. Thus, a gene encodes for a protein if transcription and translation of the mRNA corresponding to that gene in the cell or other biological system produces that protein. A "protein-encoding gene sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode for the same amino acid sequence or for amino acid sequences of essentially the same form and function.

  Polynucleotide gene sequences encoding more than one part of the expressed molecule are ligated to one another and associated regulatory sequences. For example, a functional linkage between the regulatory sequence and the heterologous nucleic acid sequence can result in expression of the latter. As another example, when the first nucleic acid sequence has a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence can be ligated with the second nucleic acid sequence. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is ligated to the coding sequence. Generally, the ligated DNA sequences are contiguous and join the coding regions in the same reading frame, where necessary and useful.

  Retroviral vectors are available (see Miller et al., 1993, Meth. Enzymol. 217: 581-599). In such an embodiment, the expressed gene is cloned into a retroviral vector for delivery to HSPC. In a particular embodiment, the retroviral vector comprises a packaging and integration of the viral genome, ie (a) a long terminal repeat (LTR) at each end of the vector, or a portion thereof, (b) a negative and Includes primers that bind sites corresponding to positive strand DNA synthesis, and (c) all cis-acting sequences required for packaging signals required for integration of genomic RNA into viral particles. For more details on retroviral vectors, see Boesen et al. , 1994, Biotherapy 6: 291-302, Clowes et al. , 1994, J .; Clin. Invest. 93: 644-651, Kiem et al. , 1994, Blood 83: 1467-1473, Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141, and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110-114. Adenovirus, adena associated virus (AAV) and alphavirus can also be used. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: 499-503, Rosenfeld et al. , 1991, Science 252: 431-434, Rosenfeld et al. , 1992, Cell 68: 143-155, Mastrangeli et al. , 1993, J .; Clin. Invest. 91: 225-234, Walsh et al. , 1993, Proc. Soc. Exp. Bioi. Med. 204: 289-300, and Lundstrom, 1999, J. Am. Recept. Signal Transduc. Res. 19: 673-686. Other gene delivery methods include mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8: 351-359), liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63: 607-618), Includes the use of ribosime (Branch and Klotman, 1998, Exp. Nephrol. 6: 78-83), and triple-stranded DNA (Chan and Glazer, 1997, J. Mol. Med. 75: 267-282).

  In a further embodiment, 70% sequence identity, 80% sequence identity, 81% sequence identity, 82% sequence identity, 83% sequence identity of any gene, protein or peptide sequence disclosed herein. Sex, 84% sequence identity, 85% sequence identity, 86% sequence identity, 87% sequence identity, 88% sequence identity, 89% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity Contains an array.

  "% Sequence identity" refers to the relationship between two or more sequences determined by comparing the sequences. It means the degree of sequence similarity between protein sequences. "Identity" (often referred to as "similarity") refers to Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988), Biocomputing: Informatics and Prod. W., ed.) Academic Press, NY (1994), Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, SJ (1994). Analysis in Molecular Biology (Von Heijne, G., e .) Academic Press 1987), and Sequence Analysis Primer (Gribskov, M. And Devereux, J., eds.) Oxford University Press, including those described NY (1992), it can be readily calculated by known methods. Preferred methods to determine sequence identity are designed to give the best match between the sequences tested. Methods to determine sequence identity and similarity are codified in published computer programs. Sequence alignments and percent identity calculations may be performed using the LASERGENE bioinformatics computing suite's Megaalign program (DNASTAR, Inc., Madison, Wisconsin). Multiple alignments of sequences also include the Clustal method of alignment with predetermined parameters (Higgins and Sharp CABIOS, 5, 151-153 (1989) (Gap penalty = 10, Gap length penalty = 10). Related programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin, AST). Altschul, et al., J. Mol. B. 215: 403-410 (1990), DNASTAR (DNASTAR, Inc., Madison, Wisconsin), and the FASTA program that incorporates the Smith-Waterman algorithm (the FASTA program-incorporating the Smith-Watermans Governance Algorithm). Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor (s): Suhai, Sandor. Publisher: Plenum, New York, NY. Within, if sequence analysis software is used for analysis, It will be appreciated that the results of the analysis of the above are based on the "default values" referenced by the program, which is any set of values or parameters that the software initially loads at initialization. means.

  Without being limited to the foregoing, proteins or peptides having sequence identity with the sequences disclosed herein include variants and D-substituted analogs thereof.

  "Variants" of the sequences disclosed herein include sequences that have one or more additions, deletions, stop positions, or substitutions as compared to the sequences disclosed herein.

  Amino acid substitutions can be conservative or non-conservative substitutions. Variants of the protein or peptide sequences disclosed herein can include those with one or more conservative amino acid substitutions. “Conservative substitution” includes substitutions found on one of the following conservative substituents. Group 1: Alanine (Ala or A), Glycine (Gly or G), Ser, Thr, Group 2: Aspartic acid (Asp or D), Glu, Group 3: Asparagine (Asn or N), Glutamine (Gln or Q) Group 4: Arg, Lysine (Lys or K), Histidine (His or H), Group 5: Ile, Leucine (Leu or L), Methionine (Met or M), Valine (Val or V), and Group 6: Phe, Tyr, Trp.

  In addition, amino acids are grouped into conservative substituents by similar function, chemical structure, or composition (eg, acidic, basic, aliphatic, aromatic, sulfur-containing). For example, the aliphatic group may include Gly, Ala, Val, Leu, and Ile depending on the purpose of the substitution. Other groups containing amino acids that are considered conservative substitutions for each other include sulfur-containing: Met and Cys, acidic: Asp, Glu, Asn, and Gln, small molecule aliphatic, non-polar or slightly polar residues. Groups: Ala, Ser, Thr, Pro, and Gly, polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln, polar, positively charged residues: His, Arg, and Lys, large molecules. Includes aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys, and large molecule aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.F. H. Found in Freeman and Company.

  "D-substituted analog" includes a protein or peptide disclosed herein having one or more L amino acids replaced with one or more D amino acids. The D amino acids can be of the same amino acid type as found in the reference sequence, or they can be different amino acids. Thus, the D analog may also be a variant.

  It is not limited to the foregoing and for illustrative purposes only.

  In certain embodiments, the binding domain is at least 80% relative to the amino acid sequence of the light chain variable region (VL) or heavy chain variable region (VH), or both, as disclosed herein. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, or 99% sequence identity, wherein each CDR is zero, or at most, from a monoclonal antibody or fragment thereof that specifically binds a cellular marker of interest. Includes one, two, or three changes.

  In certain embodiments, the binding domain is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, relative to the amino acid sequence of the TCR of the Vα, Vβ, Cα, or Cβ region. A sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, wherein And each CDR has no or at most 1, 2, or 3 alterations from them that specifically bind to the TCR or fragment or cellular marker of interest.

  In certain embodiments, the binding domain of the Vα, Vβ, Cα, or Cβ region can be derived from or based on a known TCR (eg, high affinity TCR) Vα, Vβ, Cα, or Cβ, and known. One or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (eg, 2,3,4,5,6,7,8,9,10) deletions, one or more (eg, 2,3,4,5,6,7,8,9,10) amino acid substitutions (Eg, conservative amino acid substitutions or non-conservative amino acid substitutions) or combinations of the above changes. Insertions, deletions or substitutions may be anywhere in the Vα, Vβ, Cα, or Cβ regions, including at the amino or carboxy terminus or at both ends of these regions, with each CDR unchanged or at most one, two. One or three changes may be included, and the binding domain comprising an altered Vα, Vβ, Cα, or Cβ region is capable of specifically binding its target with affinity similar to wild type. May be given.

  In certain embodiments, the binding domain VH or VL regions may be derived from or based on the VH or VL of a known monoclonal antibody, and individually or collectively as compared to the VH or VL of a known monoclonal antibody. One or more (eg 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions into one or more (eg 2, 3, 4, 5, 6, 7, 8, 9,10) deletions, one or more (eg 2,3,4,5,6,7,8,9,10) amino acid substitutions (eg conservative or non-conservative amino acid substitutions). , Or a combination of the above changes. Insertions, deletions or substitutions can be anywhere in the VH or VL regions, including at the amino or carboxy terminus or at both ends of these regions, with each CDR unchanged or at most 1, 2, or 3 It may be provided to include alterations and that the binding domain comprising the altered VH or VL region is able to bind its target more specifically with an affinity similar to the wild type binding domain.

  In certain embodiments, the binding domain is relative to the sequence of (i) scFv corresponding to FMC63, (ii) scFv corresponding to R12, (iii) scFv corresponding to R11, or (iv) scFv corresponding to Herceptin. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% sequence identity.

  In certain embodiments, the intracellular signaling domain is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% relative to CD3ζ having the sequence given in Figure 2. , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

  In certain embodiments, the costimulatory signaling domain is at least 80%, 81%, 82%, 83 relative to the intracellular domain of CD28 shown in Figure 5 or 4-1BB having the sequence given in Figure 2. %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% Can have the sequence identity of In a particular embodiment, a variant of the CD28 intracellular domain comprises an amino acid substitution at positions 186-187, wherein LL is replaced with GG.

  In certain embodiments, the transmembrane domain binds such a domain to a transmembrane domain of a similar or different surface membrane protein to minimize interaction with other members of the receptor complex. Can be chosen to avoid or can be modified by amino acid substitutions. In a further specific embodiment, the synthetic or variant transmembrane domain comprises a major hydrophobic residue such as leucine, valine. The mutant transmembrane domain preferably has a hydrophobicity index of at least 50 calculated by Kyte Doolittle. In a particular embodiment, the transmembrane domain is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with the sequence of Figure 2 or 6. It can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

  Also included are proteins and peptides having the same functionality as disclosed herein.

  Even if not expressed herein, the sequence information provided by the published databases and their knowledge possessed by those skilled in the art can be used to encode the sequences of related proteins and peptides and such proteins and peptides. Can be used to identify the gene sequence to be used.

  Differentiation. In certain embodiments, the modified HSPCs are differentiated into modified non-effector T cells prior to administration to a subject. If differentiation of the modified HSPC is required, the HSPC can be exposed to one or more growth factors that promote differentiation into non-effector T cells. This growth factor and cell culture conditions that promote differentiation are known in the art (see, eg, US Pat. No. 7,399,633, Sections 5.2 and 5.5). For example, SCF can be used in combination with GM-SCF or IL-7 to differentiate HSPC into myeloid stem / progenitor cells or lymphoid stem / progenitor cells, respectively. In certain embodiments, HSPC can be differentiated into lymphocyte stem / progenitor cells by exposing HSPC to 100 ng / ml of each of SCF and GM-SCF or IL-7. In certain embodiments, retinoic acid receptor (RAR) agonists, or preferably all-trans retinoic acid (ATRA), are used to promote the differentiation of HSPC. For example, differentiation into natural killer cells can be achieved by exposing cultured HSPC to RPMI medium supplemented with 50 U / ml of IL-2 and 500 ng / ml of IL-15 human serum. . In a further embodiment, RPMI medium can also be supplemented with L-glutamic acid.

  In certain embodiments, the modified HSPC can be differentiated into non-effector T cells including natural killer (NK) cells or neutrophils. NK cells perform two major functions. (I) recognizes and kills tumor cells and other virus-infected cells, and (ii) CCL3, CCL4, CCL5, and / or XCL1 chemokines or granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, Alternatively, the secretion of cytokines such as interferon-γ regulates innate and adaptive immune responses. Neutrophils generally circulate in the bloodstream until they reach an inflamed subject with abnormal cell types that they target and destroy.

  Compositions and formulations. The cells and modified cells can be prepared as compositions and / or formulations adapted for administration to a subject. A composition refers to a cell or modified cell prepared with a pharmaceutically acceptable carrier compatible with administration to a subject. A formulation refers to at least two cell types within a pharmaceutically acceptable carrier (hereinafter carrier) that is suitable for administration to a subject.

  Cryopreservation of cells may be necessary or beneficial at various occasions during the preparation of a composition or formulation. The terms "frozen / freezing" and "crypreserved / cryoserving" can be used interchangeably. Freezing includes freeze-drying.

  As will be appreciated by those of skill in the art, freezing cells causes freeze disruption (see Mazur, P., 1977, Cryobiology 14: 251-272), but is capable of preventing such damage. There is a means of. For example, it can be avoided by (a) use of antifreeze agents, (b) control of freezing rate, and / or (c) storage at temperatures low enough to minimize decomposition reactions. Exemplary antifreeze agents include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183: 1394-1395, Ashwood-Smith, 1961, Nature 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, 1960). , Ann. NY Acad. Sci. 85: 576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196: 548), albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D. -Mannitol (Rowe et al., 1962, Fed. Proc. 21: 157), D-sorbitol, i-inositol, D-lactose, choline chloride. (Bender et al., 1960, J. Appl. Physiol. 5: 520), amino acids (Phan The Tran and Bender, 1960, Exp. Cell Res. 20: 651), methanol, acetamide, glycerol monoacetate (Lovelock, 1954). , Biochem. J. 56: 265), and inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104: 388, Phan The Tran and Bender, 1961, radiation biology, radiation biology. Proceedings of the Third Australian Conference (in Radiobiology, Procedings of the Third Australian Conference) on Radiobiology,) Ilbury ed., Butterworth, London, p. 59). In certain embodiments, DMSO can be used. Addition of plasma (eg up to 20-25% concentration) can enhance the protective effect of DMSO. Since 1% DMSO concentration is toxic at temperatures above 4 ° C, cells can be kept at 0 ° C until freezing after addition of DMSO.

  In the cryopreservation of cells, slow cooling rate control is important and different cryoprotectants (Rapatz et al., 1968, Cryobiology 5 (1): 18-25) and different cell types have different optimal cooling rates. (For example, Rowe and Rinfret, 1962, Blood 20: 636, Rowe, 1966, Cryobiology 3 (1): 12-18, Lewis, et al., 1967, Transfusion 7 (1): 17-32, and stem cells. See Mazur, 1970, Science 168: 939-949 for the effect of cooling rate on their survival and their transplantation potential). The hot-melt phase where water becomes ice should be minimized. The cooling means can be implemented, for example, by utilizing a programmable freezer or methanol bath procedure. The programmable freezer allows determination of optimal cooling rates and promotion of standard regenerative cooling.

  In certain embodiments, DMSO-treated cells are transferred to trays containing chilled methanol that can be pre-chilled on ice and are placed side-by-side in a -80 ° C mechanical refrigerator (eg, Harris or Revco). You can For thermocouple measurements of methanol baths and samples, thermocouples with cooling rates of 1 ° C to 3 ° C / min are recommended. After at least 2 hours, the specimen reached -80 ° C and can be placed directly in liquid nitrogen (-196 ° C).

  After complete freezing, the cells can be rapidly transferred to long term cryogenic storage vessels. In a preferred embodiment, the samples can be stored in liquid nitrogen (-196 ° C) or nitrogen vapor (-1 ° C) at cryogenic temperatures. Such storage is facilitated by the availability of high efficiency liquid nitrogen freezer.

  Further considerations and procedures for cell manipulation, cryopreservation and long term storage can be found in the exemplary references below. U.S. Pat. Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107-186, Livesey and Linner, 1987, Nature 327: 255, Linner et al. , 1986, J. Am. Histochem. Cytochem. 34 (9): 1123-1135, Simione, 1992, J. Am. Parenter. Sci. Technol. 46 (6): 226-32).

  The following frozen storage and frozen cells can be thawed according to their use according to methods known to those skilled in the art. Preferably, frozen cells are thawed immediately and refrigerated immediately upon thawing. In certain embodiments, the vial containing the frozen cells is soaked in a warm water bath up to its neck. Gentle rotation will thaw the cells and enhance heat transfer from their warm water to the inner ice portion, ensuring mixing of the cell suspension. As soon as the ice is completely melted, the vial can be placed on the ice immediately.

  In certain embodiments, steps can be taken to prevent cell aggregation upon thawing. Exemplary methods include low molecular weight dextran and citric acid, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20: 17-24), before and / or after freezing of DNase. Addition (Spitzer et al., 1980, Cancer 45: 3075-3085).

  As will be appreciated by one of skill in the art, if a cryoprotectant that is toxic to humans is used, then the cryoprotectant should be removed prior to therapeutic use. DMSO has no serious toxicity.

  Exemplary cell administration carriers and methods are described on pages 14-15 of US Patent Publication No. 2010/0183564. Additional pharmaceutical carriers are described in Remington. The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed. , Lippicott Williams & Wilkins (2005).

  In certain embodiments, cells can be harvested from the medium and washed and concentrated in a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, saline, water, Hanks solution, Ringer's solution, Nonnosol-R (Abbott Labs), Plasma-Lyte A® (Baxter Laboratories, Inc., Morton). Grove, IL), glycerol, ethanol, and combinations thereof.

  In certain embodiments, the carrier can be supplemented with human serum albumin (HSA) or other human serum components or fetal calf serum. In certain embodiments, the infusion carrier comprises 5% HAS or glucose buffered saline. Further isotonic agents include polyhydric sugar alcohols including trihydric or higher polyhydric sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

  The carrier may be a buffer such as citrate buffer, succinate buffer, tartaric acid buffer, fumaric acid buffer, gluconate buffer, oxalate buffer, buffer lactic acid, acetate buffer, phosphate buffer, histidine buffer and / or trimethylamine salt. Can be included.

  Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives that help prevent cell attachment to the walls of containers. Typical stabilizing agents include polyhydric sugar alcohols, amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine, lactose, trehalose, Organic sugars or sugar alcohols such as stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol and cyclitols such as inositol, PEG, amino acid polymers, urea, glutathione, thioctic acid, sodium thioglycolate, thio. Glycerol, α-monothioglycerol, and sulfur-containing reducing agents such as sodium thiosulfate, low molecular weight polypeptides (ie <10 residues), HSA, bovine serum albumin, Proteins such as ratin or immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, monosaccharides such as xylose, mannose, fructose and glucose, disaccharides such as lactose, maltose and sucrose, trisaccharides such as raffinose, and polysaccharides such as dextran. Sugars can be included.

  If necessary or beneficial, the composition or formulation can include a local anesthetic such as lidocaine that relieves pain at the site of injection.

  Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halide, hexamethonium chloride, methyl, or alkylparabens such as propylparaben, Includes catechol, resorcinol, cyclohexanol, and 3-pentanol.

The amount of therapeutically effective cells in the composition or formulation is greater than 10 ² cells, greater than 10 ³ cells, greater than 10 ⁴ cells, greater than 10 ⁵ cells, greater than 10 ⁵ cells, 10 ^There may be more than ⁶ cells, more than 10 ⁷ cells, more than 10 ⁸ cells, more than 10 ⁹ cells, more than 10 ¹⁰ cells, or more than 10 ¹¹ cells.

In the compositions and formulations disclosed herein, cells are generally in a volume of 1 liter or less, 500 mls or less, 250 mls or less, or 100 mls or less. Thus, the density of cells administered is usually higher than 10 ⁴ cells / ml, 10 ⁷ cells / ml or 10 ⁸ cells / ml.

  As indicated, the composition comprises one cell type (eg, modified HSPC or modified effector). The formulation includes HSPC, modified HSPC and / or modified effectors (such as modified NK cells) in combination. In certain embodiments, modified HSPCs and modified effectors of the same binding domain are combined. In other embodiments, different binding domain modified HSPCs and modified effectors are combined. Similarly, all other aspects of an expressed molecule (eg, effector domain component, spacer region, etc.) can be the same or different in various combinations between modified HSPCs and modified effectors in a formulation. . Furthermore, modified HSPCs or their components that express different molecules can be included together in the formulation, and modified effectors that express different molecules or their components can be included together in the formulation. In certain embodiments, one formulation can include at least two modified HSPCs expressing different molecules and at least two modified effector cells expressing different molecules.

  HSPC, modified HSPC and modified effector are, for example, a 1: 1 to 1 ratio, a 2: 1 to 1 ratio, a 1 to 2: 1 ratio, a 1: 1 to 2 ratio, a 5: 1 to 1 ratio. 1 to 5 to 1 ratio, 1 to 1 to 5 ratio, 10 to 1 to 1 ratio, 1 to 10 to 1 ratio, 1 to 1 to 10 ratio, 2 to 2 to 1 ratio, 1 2 to 2 ratio, 2 to 1 to 2 ratio, 5 to 5 to 1 ratio, 1 to 5 to 5 ratio, 5 to 1 to 5 ratio, 10 to 10 to 1 ratio, 1 to 10 It can be combined in different ratios such as a ratio of 10 to 10, a ratio of 1 to 10 and the like. These ratios can also be adapted to the number of cells expressing the same or different components of the molecule. If only two cell types are combined or only two combinations of expressed molecular components are included in the formulation, the ratio includes any two-digit combination that can be made from the three-digit combination described above. be able to. In embodiments, the combined cell populations are tested for in vivo and / or in vitro efficacy and / or cell proliferation, and the ratio of cells provided to efficacy and / or cell proliferation is selected. To be done.

  The compositions and formulations disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion, or lavage. The compositions and formulations further include bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic and nodular, intraperitoneal, intralesional, intraprostatic, vaginal, rectal, topical, intrathecal, tumor. It can be prescribed for intramuscular, intramuscular, intravesical and / or subcutaneous injection.

  kit. The kit can include one or more cells of interest, one or more containers containing the compositions or formulations described herein. In certain embodiments, the kit comprises one or more cells, a composition or formulation and / or one or more containers containing a composition for use in combination with another cell, composition or formulation. Can be included. Such container-related matters shall be notified in a prescribed format established by a governmental agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, which should be manufactured and used for human administration. Or reflects approval by the agency responsible for the sale. The notification will state that the donor cells, compositions or formulations can be administered to a subject without immunocompatibility. The kit can include instructions for using the kit, such as, for example, instructions on the preparation of cells, compositions and / or formulations for administration, proper disposal of associated waste, and the like. The instructions can be in the form of printed material provided in the kit, or the instructions can be printed on a portion of the kit itself. The instructions may be in the form of procedures, brochures, booklets, CD-Roms, computer readable devices, or may provide instructions for using the instructions from a remote location such as a website. In certain embodiments, the kits also effectively use the kits, such as syringes, ampoules, tubes, face masks, needle-free fluid delivery devices, injection caps, sponges, sterile adhesive strips, Chloraprep, gloves, and the like. It may include some or all of the medical supplies needed to do so. Modifications can be made in the contents of any of the kits described herein.

  how to use. The methods disclosed herein include subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cows, goats, pigs) treated with the cells disclosed herein. , Chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.)). The subject to be treated includes delivery of a therapeutically effective amount. A therapeutically effective amount includes an effective amount, a prophylactic and / or therapeutic treatment is given.

  An "effective amount" is the number of cells needed to bring about a desired physiological change in a subject. Effective amounts are often administered for research purposes. An effective amount disclosed herein provides (i) blood support by reducing immunodeficiency, pancytopenia, neutropenia and / or leukopenia (eg, repopulating cells of the immune system). Giving and (ii) having an anti-cancer effect.

  “Prophylactic treatment” is a treatment that is administered to a subject who does not exhibit the signs or symptoms of the condition to be treated or who has the initial signs or symptoms of the condition to be treated, which gradually reduces the risk of developing the condition, Including cases where treatment is provided for the purpose of preventing or reducing. Therefore, prophylactic treatment functions as a prophylactic treatment for a condition.

  “Therapeutic treatment” includes treatment administered to a subject who is exhibiting symptoms or signs of a condition, and to that subject for the purpose of reducing the severity or progression of the condition. Is administered.

  The actual dose administered to a particular subject can be determined by, for example, physical and physiological factors including the target, weight, type of condition, severity of condition, expected related events, if known, previous or concurrent events. Can be determined by a physician, veterinarian, or researcher considering parameters such as the therapeutic intervention, the idiopathic disease of the subject, and the route of administration. In addition, in vivo and in vitro assays can optionally be incorporated to help identify optimal dosage ranges.

The therapeutically effective amount to be administered is above 10 ² cells, above 10 ³ cells, above 10 ⁴ cells, above 10 ⁵ cells, above 10 ⁶ cells, above 10 ⁷ cells, 10 ^More than ⁸ cells, more than 10 ⁹ cells, more than 10 ¹⁰ cells, or more than 10 ¹¹ can be included.

  As indicated, the compositions and formulations disclosed herein can be administered by, for example, injection, infusion, perfusion, or lavage, and more particularly, bone marrow, intravenous, intradermal, intraarterial, Intranodal, intralymphatic and nodular, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, and / or subcutaneous and / or bolus injection Administration may be via one or more.

  The use of unmodified HSPC is described in US Pat. No. 7,399,633, section 5.6.1 and WO 2013/086436. HSPCs and modified HSPCs can be administered for similar or different purposes. The common purpose is to give the subject a hematopoietic function as needed, and / or immunodeficiency, pancytopenia, neutropenia and / or leukopenia (periodic neutropenia and idiopathic Treatment of one or more of these (including neutropenia) (collectively “purpose”). The HSPCs and modified HSPCs can be administered to a subject having a reduced blood cell level or at risk of developing a reduced blood cell level relative to a control blood cell level. In certain embodiments, the subject has or is at risk of developing anemia.

  Treatments for this purpose include alkylating agents, cytarabine (Ara-C), azathioprine, carboplatin, cisplatin, chlorambucil, clofarabine, cyclophosphamide, ifosfamide, mechlorethamine, mercaptopurine, oxaliplatin, taxanes, and vinca alkaloids. For example, it may need to be based on exposure to intensive chemotherapy, including exposure to one or more of vincristine, vinblastine, vinorelbine, and vindesine).

  Treatment for that purpose may also need to be based on exposure to myeloablative therapy in response to hematopoietic cell transplantation (HCT). In certain embodiments, HSPCs and / or modified HSPCs are administered to bone marrow donors at risk of degrading bone marrow or at risk of developing depleted or limited blood cell levels. Administration can occur before or after bone marrow acquisition. HSPCs and / or modified HSPCs can also be administered to recipients of bone marrow transplants.

  Treatments for that purpose also include exposure to acute ionizing radiation and / or antibiotics, penicillin, gancyclovir, daunomycin, sulfa drugs, phenothiazines, tranquilizers, meprobamate, analgesics, aminopyrine, dipyrone, anticonvulsants, phenytoin, carbamazepine, It may need to be based on exposure to myelosuppressive or other agents that may cause hematopoietic defects, including antithyroid drugs, propylthiouracil, methimazole, and diuretics.

  Treatment for that purpose should also be based on the result of treatment for a virus (eg, HIVI, HIVII, HTLVI, HTVII, HTVIII), microbial or parasite infection and / or renal disease or renal failure, eg dialysis. possible. For example, multiple immunodeficiencies in T and / or B lymphocytes, or immune disorders such as rheumatoid arthritis, may also be beneficially affected by treatment with HSPC and / or modified HSPC. Immunodeficiency may also be the result of another medical procedure.

  HSPCs and / or modified HSPCs can also be used to treat aplastic anemia, Chediak-East syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome, myelofibrosis or thrombocytopenia. Severe thrombocytopenia also results from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May-Hegglin's syndrome. Acquired thrombocytopenia also results from autologous or alloantibodies, such as in immune thrombocytopenic purpura, systemic lupus erythromatosis, hemolytic anemia, or fetal maternal incompatibility. In addition, splenomegaly, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, infections, and / or prosthetic heart valves lead to thrombocytopenia. Thrombocytopenia also results from bone marrow infiltration by cancer, lymphoma, leukemia or fibrosis.

  In certain embodiments, the subject has or is at risk of bleeding, eg, due to trauma. In certain embodiments, the subject suffers from depleted bone marrow associated with, for example, a congenital, genetic or acquired syndrome characterized by bone marrow loss or depleted bone marrow. In certain embodiments, the subject is in need of hematopoiesis.

  Administration of HSPC or modified HSPC to a subject, as shown in connection with bone marrow donors, can occur at any time during the treatment regimen that is considered useful by the therapist. As a non-limiting example, HSPCs and / or modified HSPCs can be administered to a subject prior to, concurrently with, or following, for example, chemotherapy, radiation treatment or bone marrow transplant. HSPCs and / or modified HSPCs are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 (or 1, 2 ,, or 10 days after administration of the HSPCs and / or modified HSPCs to a subject. More than or less than 3, 4, 5, 6, 7, 8, 9, 10 days), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks (or 1, 2,3,4,5,6,7,8,9,10 weeks or more), 1,2,3,4,5,6,7,8,9,10,11,12 Months (or more than or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months) 1, 2, 3, 4, 5 years When assayed (or over 1, 2, 3, 4, 5 years, or less), it can effectively engraft. In certain embodiments, said HSPC and / or modified HSPC is within 10 days, within 2 weeks, within 3 weeks, within 4 weeks, within 6 weeks after administration of said HSPC and / or CAR-HSPC to a subject. , Or is assayed within 13 weeks, it is effective in providing engraftment.

  HSPC, modified HSPC and modified effectors. HSPCs, modified HSPCs and modified effectors can be administered for different purposes within a treatment regimen. The use of HSPCs and modified HSPCs to provide blood support and modified HSPCs and modified effectors that confer a graft effect on leukemia in all treatments are described above. Similar approaches can be used to provide blood support and / or target unwanted cancer cells and as an adjunct treatment to chemotherapy or radiation therapy.

  Exemplary cancers that can be treated with modified HSPC and modified effectors include adrenal gland cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cancer, cervical cancer, colon cancer, Colorectal cancer, corpus uterine cancer, ear, nose and throat (ENT) cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, Hodgkin's disease, intestinal cancer, kidney Cancer, laryngeal cancer, leukemia, liver cancer, lymph node cancer, malignant lymphoma, lung cancer, melanoma, mesothelioma, myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, Ovarian cancer, pancreatic cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, sarcoma, seminoma, skin cancer, stomach cancer, malformation, testicular cancer, thyroid cancer, uterine cancer, vagina Cancer, vascular tumors, and their metastases.

  In the context of cancer, a therapeutically effective amount has an anti-cancer effect. Anti-cancer effects include reduction in tumor cell number, reduction in metastasis, reduction in tumor burden, increase in lifespan, induction of cancer cell apoptosis, induction of cancer cell death, suppression of cancer cell growth, tumor Growth inhibition, prevention of metastasis, prolongation of the subject's life, and / or reduction of recurrence or recurrence of cancer after treatment can be quantified.

  In the context of blood support, a therapeutically effective amount treats immunodeficiency, pancytopenia, neutropenia and / or leukopenia by increasing the desired number of cells in the subject's circulation. By increasing the number of immune system cells and / or immune system cell precursors, the desired number of cells in the subject's circulation can be increased to repopulate the subject's immune system.

  In certain embodiments that utilize modified HSPCs and modified effectors, cancer cells of a subject can be characterized by the presence of cellular markers. Binding domains expressed by modified HSPCs or modified effectors can be selected based on the characteristics of their cellular markers. In certain embodiments, pre-produced modified HSPCs and modified effectors are selected based on their ability to bind cell markers selectively expressed on cancer cells of a particular subject.

  When formulated to treat cancer, the compositions and formulations of the present disclosure also include p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, Bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, TNF-α, and / or HSV-tk. Plasmid DNA carrying one or more anti-cancer genes selected from The compositions and formulations also include adriamycin, angiostatin, azathioprine, bleomycin, busulfan, camptothecin, carboplatin, carmustine, chlorambucil, chlormetheamine, chloroquinoxaline, sulfonamide, cisplatin, cyclophosphamide, cycloplatam, cytarabine, dacarbazine, dactino. Mycin, daunorubicin, didox, doxorubicin, endostatin, enloplatin, estramustine, etoposide, extramustine phosphate, flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate, hydroxyurea, idoxuridine, interferon, interleukin, interleukin. , Leuprolide, lobaplatin, ro Stin, mannomustine, mechlorethamine, mechlorethamine oxide, melphalan, mercaptopurine, methotrexate, mithramycin, mitobronitol, mitomycin, mycophenolic acid, nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine, platinum-triamine complex, prikamycin, prednisolone. , Prednisone, procarbazine, protein kinase C inhibitor, puromycin, semustine, signal transduction inhibitor, spiroplatin, streptozotocin, stromelysin inhibitor, taxol, tegafur, telomerase inhibitor, teniposide, thalidomide, thiamiprine, thioguanine, thiotepa, thiamiprine, Tretamine, triazicon, trofosfamide (trifosf) mide), tyrosine kinase inhibitors, uramustine, vidarabine, vinblastine, vinca alkaloids, vincristine, vindesine, vorozole, Zenipurachin, including Zenipurachin or zinostatin may be obtained by or administered a combination of one or more anti-cancer agents.

  Modified HSPC and modified effectors. Modified HSPCs and / or modified effectors are used to confer hematopoietic function, or for the treatment of immunodeficiency, pancytopenia, neutropenia and / or leukopenia, where treatment is undesirable or not necessary. Can be used without HSPC.

  As will be appreciated by those in the art, different animal models of blood disorders and cancers, if known and necessary or beneficial, can be used to assess the effectiveness of a particular therapeutic framework.

  The following examples and exemplary embodiments are included for the purpose of representing specific embodiments of the present disclosure. Those skilled in the art will appreciate that numerous changes can be made to the particular embodiments disclosed herein and that similar or similar results can be obtained without departing from the spirit and scope of the disclosure. , Should be recognized in light of this disclosure.

Exemplary embodiment.
1. (I) an extracellular component containing a ligand binding domain that binds CD19, (ii) an intracellular component containing an effector domain containing the cytoplasmic domain of CD28 or 4-1BB, (iii) a spacer region containing the hinge region of human IgG4, And (iv) a CD34 + hematopoietic stem progenitor cell (HSPC) that has been genetically modified to express the human CD4 or CD28 transmembrane domain.
2. The said ligand binding domain is RASQDISKYLN (SEQ ID NO.108) CDRL1 sequence, SRLHSGV (SEQ ID NO.111) CDRL2 sequence, GNTLPYTFG (SEQ ID NO.104) CDRL3 sequence, DYGVS (SEQ ID NO.103). CDRH1 sequence of V I The HSPC according to embodiment 1, which is a single-chain Fv fragment (scFv) comprising a CDRH2 sequence of WGSETTYNYNSALKS (SEQ ID NO. 114) and a CDRH3 sequence of YAMDYWG (SEQ ID NO. 115).
3. The HSPC according to embodiment 1 or 2, wherein the spacer region is 12 or less amino acids.
4. The HSPC according to any one of embodiments 1-3, wherein the spacer region comprises SEQ ID NO: 47.
5. (I) an extracellular component containing a ligand binding domain that binds CD19, (ii) an intracellular component containing an effector domain containing the cytoplasmic domain of CD28 or 4-1BB, (iii) a spacer region containing the hinge region of human IgG4, And (iv) non-effector T cells genetically modified to express human CD4 or CD28 transmembrane domain.
6. The ligand binding domain is RASQDISKYLN (SEQ ID NO. 108) CDRL1 sequence, SRLHSGV (SEQ ID NO. 111) CDRL2 sequence, GNTLPYTFG (SEQ ID NO. 104) CDRL3 sequence, DYGVS (SEQ ID NO. 103). CDRH1 sequence of V I The non-effector T cell according to embodiment 5, which is a single-chain Fv fragment (scFv) comprising a CDRH2 sequence of WGSETTYNYNSALKS (SEQ ID NO. 114) and a CDRH3 sequence of YAMDYWG (SEQ ID NO. 115).
7. The non-effector T cell according to embodiment 5 or 6, wherein the spacer region is 12 or less amino acids.
8. The non-effector T cell of any one of embodiments 5-7, wherein the spacer region comprises SEQ ID NO: 47.
9. The non-effector T cell according to any one of embodiments 5-8, wherein the non-effector T cell is a natural killer cell.
10. SEQ ID NOs: 34, 53, 54, 55, 56, 57, or 58 chimeric antigen receptors (CARs) that have been genetically modified to express hematopoietic stem progenitor cells (HSPCs).
11. The HSPC according to embodiment 10, wherein the HSPC is CD34 +.
12. SEQ ID NO: 34, 53, 54, 55, 56, 57, or 58 CAR non-effector genetically modified to express a CAR.
13. The non-effector T cell according to embodiment 12, wherein the non-effector T cell is a natural killer cell.
14. Genetically modified to express (i) an extracellular component containing a ligand-binding domain that binds a cell marker selectively expressed on unwanted cells, and (ii) an intracellular component containing an effector domain HSPC.
15. The HSPC of embodiment 14, wherein the ligand binding domain is an antibody fragment.
16. 16. The HSPC of embodiment 14 or 15, wherein the ligand binding domain is a single chain variant fragment of an antibody.
17. The HSPC of any one of embodiments 14-16, wherein the ligand binding domain binds CD19.
18. The said ligand binding domain is RASQDISKYLN (SEQ ID NO.108) CDRL1 sequence, SRLHSGV (SEQ ID NO.111) CDRL2 sequence, GNTLPYTFG (SEQ ID NO.104) CDRL3 sequence, DYGVS (SEQ ID NO.103). CDRH1 sequence of V I The HSPC according to any one of embodiments 14-17, which is an scFv comprising the CDRH2 sequence of WGSETTYNYNSALKS (SEQ ID NO.114) and the CDRH3 sequence of YAMDYWG (SEQ ID NO.115).
19. 19. The HSPC of embodiment 18, wherein the HSPC has also been genetically modified to express a spacer region of 12 amino acids or less.
20. The HSPC of embodiment 19, wherein the spacer region comprises SEQ ID NO: 47.
21. The HSPC of any one of embodiments 14-16, wherein the ligand binding domain binds ROR1.
22. The ligand binding domain is a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 101), a CDRL2 sequence of TIYPSSG (SEQ ID NO. 112), a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 100), DTIDWY (SEQ ID NO. 102). 22. HSPC according to any one of embodiments 14-16 or 21, which is an scFv comprising the CDRH1 sequence of VQSDGSYTKRPPGVPDR (SEQ ID NO.113) and the CDRH3 sequence of YIGGYVFG (SEQ ID NO.117). .
23. The ligand-binding domain is a CDRL1 sequence of SGSDINDYPIS (SEQ ID NO.109), a CDRL2 sequence of INSGGST (SEQ ID NO.105), a CDRL3 sequence of YFCARGYS (SEQ ID NO.116), and SNLAW (SEQ ID NO.110). HSPC according to any one of embodiments 14-16 or 21, which is a scFv comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of RASNLASGVPSRFSGS (SEQ ID NO. 107), and the CDRH3 sequence of NVSYRTSF (SEQ ID NO. 106). .
24. 24. The HSPC of embodiment 23, wherein the HSPC is also genetically modified to express a spacer region of 229 or less amino acids.
25. The HSPC of embodiment 24, wherein the spacer region comprises SEQ ID NO: 61.
26. The HSPC according to any one of embodiments 14-16, wherein the ligand binding domain binds PSMA, PSCA, mesothelin, CD20, WT1, or Her2.
27. The intracellular component is 4-1BB, CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, LAG3, LAT, Lck, LRP, One or more signal transduction and / or stimulation domains selected from NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, and Zap70 signaling and / or stimulation domains. HSPC according to any one of embodiments 14-26, which comprises an effector domain containing
28. The HSPC according to any one of embodiments 14-27, wherein said intracellular component comprises an effector domain containing an intracellular signaling domain of CD3ζ, CD28ζ, or 4-1BB.
29. The intracellular component is from the costimulatory domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, lymphocyte function-related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3. The HSPC according to any one of embodiments 14-28, which comprises an effector domain containing one or more co-stimulatory domains selected.
30. The intracellular component is (i) all or part of the signal transduction domain of CD3ζ, (ii) all or part of the signal transduction domain of CD28, (iii) all or part of the signal transduction domain of 4-1BB. Any of Embodiments 14-29, which comprises a portion, or (iv) an effector domain containing an intracellular signaling domain that comprises all or part of the signaling domain of CD3ζ, CD28, and / or 4-1BB. HSPC according to one.
31. The HSPC according to any one of embodiments 14-30, wherein said intracellular component comprises a variant of CD3ζ and / or an effector domain containing a portion of the intracellular signaling domain of 4-1BB.
32. The HSPC of any one of embodiments 14-18, 21-23, or 26-31, wherein the HSPC is also genetically modified to express a spacer region.
33. The HSPC of embodiment 32, wherein the spacer region comprises a portion of the hinge region of a human antibody.
34. 34. The HSPC of embodiment 32 or 33, wherein the spacer region comprises a hinge region and at least one other portion of the Fc domain of a human antibody selected from CH1, CH2, CH3 or combinations thereof.
35. The HSPC according to embodiment 32 or 33, wherein the spacer region comprises an Fc domain and a human IgG4 heavy chain hinge.
36. The HSPC according to embodiment 32, wherein the spacer region is a region having a length selected from 12 or less amino acids, 119 or less amino acids, or 229 or less amino acids.
37. The HSPC according to embodiment 32, wherein the spacer region is SEQ ID NO: 47, SEQ ID NO: 52, or SEQ ID NO: 61.
38. The HSPC according to any one of embodiments 14-37, wherein the HSPC is also genetically modified to express a transmembrane domain.
39. The HSPC according to embodiment 38, wherein said transmembrane domain is a CD28 transmembrane domain or a CD4 transmembrane domain.
40. The HSPC according to any one of embodiments 14-39, wherein said extracellular component further comprises a tag sequence.
41. The HSPC according to embodiment 40, wherein the tag sequence is EGFR lacking an intracellular signaling domain.
42. The HSPC according to any one of embodiments 14-41, wherein the HSPC is CD34 +.
43. A non-effector T cell that has been genetically modified to express (i) an extracellular component that includes a ligand binding domain that binds a cellular marker on an unwanted cell, and (ii) an intracellular component that includes an effector domain.
44. The non-effector T cell of embodiment 43, wherein the ligand binding domain is an antibody fragment.
45. The non-effector T cell of embodiment 43 or 44, wherein said ligand binding domain is a single chain variable fragment of an antibody.
46. The non-effector T cell of any one of embodiments 43-45, wherein said ligand binding domain binds CD19.
47. The said ligand binding domain is RASQDISKYLN (SEQ ID NO.108) CDRL1 sequence, SRLHSGV (SEQ ID NO.111) CDRL2 sequence, GNTLPYTFG (SEQ ID NO.104) CDRL3 sequence, DYGVS (SEQ ID NO.103). CDRH1 sequence of V I 47. The non-effector T cell of any one of embodiments 43-46, which is an scFv comprising the CDRH2 sequence of WGSETTYNYSALKS (SEQ ID NO. 114) and the CDRH3 sequence of YAMDYWG (SEQ ID NO. 115).
48. The non-effector T cell of embodiment 47, wherein said non-effector T cell is also genetically modified to express a spacer region of 12 or less amino acids.
49. The non-effector T cell of embodiment 48, wherein the spacer region comprises SEQ ID NO: 47.
50. The non-effector T cell of any one of embodiments 43-45, wherein said ligand binding domain binds ROR1.
51. The ligand binding domain is a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 101), a CDRL2 sequence of TIYPSSG (SEQ ID NO. 112), a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 100), DTIDWY (SEQ ID NO. 102). Non-human according to any one of embodiments 43-45 or 50, which is a scFv comprising a CDRH1 sequence of VQSDGSYTKRPPGVPDR (SEQ ID NO. 113) and a CDRH3 sequence of YIGGYVFG (SEQ ID NO. 117). Effector T cells.
52. The ligand-binding domain is a CDRL1 sequence of SGSDINDYPIS (SEQ ID NO.109), a CDRL2 sequence of INSGGST (SEQ ID NO.105), a CDRL3 sequence of YFCARGYS (SEQ ID NO.116), and SNLAW (SEQ ID NO.110). Any of embodiments 43-45 or 50, which is a single chain Fv fragment (scFv) comprising the CDRH1 sequence of SEQ ID NO: 1, the CDRH2 sequence of RASNLASGVPSRFSGS (SEQ ID NO. 107), and the CDRH3 sequence of NVSYRTSF (SEQ ID NO. 106). Non-effector T cells according to one of the items 1.
53. 53. The non-effector T cell of embodiment 52, wherein the non-effector T cell is also genetically modified to express a spacer region of 229 or less amino acids.
54. The non-effector T cell of embodiment 53, wherein the spacer region comprises SEQ ID NO: 61.
55. The non-effector T cell of any one of embodiments 43-45, wherein the ligand binding domain binds PSMA, PSCA, mesothelin, CD20, WT1, or Her2.
56. The intracellular component is 4-1BB, CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, LAG3, LAT, Lck, LRP, One or more signal transduction and / or stimulation domains selected from NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, and Zap70 signaling and / or stimulation domains. A non-effector T cell according to any one of embodiments 43-55, which comprises an effector domain containing
57. 57. The non-effector T cell of any one of embodiments 43-56, wherein said intracellular component comprises an effector domain containing an intracellular signaling domain of CD3ζ, CD28ζ, or 4-1BB.
58. The intracellular component is one or more costimulatory domains selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, LFA-1, CD2, CD7, LIGHT, NKG2C, or B7-H3 costimulatory domains. The non-effector T cell of any one of embodiments 43-57, which comprises an effector domain containing
59. The intracellular component is (i) all or part of the signal transduction domain of CD3ζ, (ii) all or part of the signal transduction domain of CD28, (iii) all or part of the signal transduction domain of 4-1BB. Any of embodiments 43-58, which comprises a portion, or (iv) an effector domain containing an intracellular signaling domain comprising all or part of the signaling domain of CD3ζ, CD28, and / or 4-1BB. The non-effector T cell according to one.
60. The non-effector T cell of any one of embodiments 43-59, wherein said intracellular component comprises a variant of CD3ζ and / or an effector domain containing a portion of the intracellular signaling domain of 4-1BB. .
61. The non-effector T cell of any one of embodiments 43-47, 50-52, or 55-60, which is genetically modified to express a spacer region.
62. 62. The non-effector T cell of embodiment 61, wherein the spacer region comprises a portion of the hinge region of a human antibody.
63. 63. The non-effector T cell of embodiment 61 or 62, wherein the spacer region comprises a hinge region and at least one other portion of the Fc domain of a human antibody selected from CH1, CH2, CH3 or combinations thereof.
64. 63. The non-effector T cell of embodiment 61 or 62, wherein the spacer region comprises an Fc domain and a human IgG4 heavy chain hinge.
65. The non-effector T cell of embodiment 61, wherein the spacer region is a region having a length selected from 12 or less amino acids, 119 or less amino acids, or 229 or less amino acids.
66. 62. The non-effector T cell of embodiment 61, wherein the spacer region is SEQ ID NO: 47, SEQ ID NO: 52, or SEQ ID NO: 61.
67. The non-effector T cell of any one of embodiments 43-66, wherein said non-effector T cell has also been genetically modified to express a transmembrane domain.
68. The non-effector T cell of embodiment 67, wherein the transmembrane domain is a CD28 transmembrane domain or a CD4 transmembrane domain.
69. The non-effector T cell of any one of embodiments 43-68, wherein said extracellular component further comprises a tag sequence.
70. The non-effector T cell of embodiment 69, wherein the tag sequence is EGFR lacking an intracellular signaling domain.
71. The non-effector T cell of any one of embodiments 43-70, wherein said non-effector T cell is a natural killer cell.
72. A composition comprising a genetically modified HSPC according to any one of embodiments 1-4, 10, 11 or 14-42.
73. A composition comprising non-effector T cells according to any one of embodiments 5-9, 12, 13, or 43-71.
74. The composition according to embodiment 72 or 73, formulated for infusion or injection.
75. A formulation comprising HSPC and a genetically modified HSPC according to any one of embodiments 1-4, 10, 11 or 14-42.
76. A formulation comprising HSPC and a genetically modified non-effector T cell according to any one of embodiments 5-9, 12, 13, or 43-71.
77. A genetically modified HSPC according to any one of embodiments 1-4, 10, 11 or 14-42 and any one of embodiments 5-9, 12, 13 or 43-71. A formulation comprising non-effector T cells according to claim 1.
78. The formulation of embodiment 77, further comprising HSPC.
79. The formulation of any one of embodiments 75-78, formulated for infusion or injection.
80. A kit comprising the composition of any one of embodiments 72-74, comprising instructions advising that the composition or formulation can be administered to a subject without immunological compatibility.
81. A kit comprising the formulation of any one of embodiments 75-79, including instructions advising that the composition or formulation can be administered to a subject without immunocompatibility.
82. The composition according to any one of embodiments 72-74 and embodiments 75-79, including instructions advising that the composition or formulation can be administered to a subject without immunological compatibility. A kit comprising the formulation of any one.
83. A cell comprising administering to a subject a therapeutically effective amount of a genetically modified HSPC, the genetically modified HSPC being (i) selectively expressed on unwanted cancer cells. Expressing an extracellular component containing a ligand binding domain that binds a marker and (ii) an intracellular component containing an effector domain, thereby repopulating the subject's immune system and targeting unwanted cancer cells , A method of repopulating the immune system of a subject in need thereof and targeting unwanted cancer cells in the subject.
84. The method of embodiment 83, further comprising administering genetically modified non-effector T cells to the subject, wherein the genetically modified non-effector T cells are (i) undesired. The aforementioned method, wherein an extracellular component containing a ligand binding domain and (ii) an intracellular component containing an effector domain, which binds a cell marker selectively expressed on cancer cells, are expressed.
85. 85. The method of embodiment 83 or 84, further comprising administering HSPC.
86. The method according to any one of embodiments 83-85, wherein no immunological adaptation to the subject is required prior to administration.
87. 87. The method of any one of embodiments 83-86, wherein the cell marker is CD19, ROR1, PSMA, PSCA, mesothelin, CD20, WT1, or Her2.
88. Proliferation needs to be based on exposure to myeloablative therapy in response to hematopoietic cell transplantation (HCT) and the unwanted cancer cells are CD19-expressing acute lymphoblastic leukemia cells , The method of any one of embodiments 83-87.
89. The method according to any one of embodiments 83-88, wherein said subject is a patient with recurrent childhood acute lymphoblastic leukemia.
90. A ligand that administers to a subject a therapeutically effective amount of a genetically modified non-effector T cell, the genetically modified non-effector T cell binding to (i) a selectively expressed cell marker. Expressing an extracellular component containing a binding domain, and (ii) an intracellular component containing an effector domain, and identifying at least one cell marker selectively expressed on cancer cells from the subject. A method of targeting unwanted cancer cells in a subject.
91. Administering a genetically modified HSPC to a subject, wherein the genetically modified HSPC comprises (i) an extracellular component comprising a ligand binding domain that binds a selectively expressed cell marker, and (Ii) The method of embodiment 90, which expresses an intracellular component that includes an effector domain.
92. A method for targeting unwanted cancer cells in a subject, comprising identifying at least one cell marker selectively expressed on cancer cells from the subject, the genetically modified HSPC. Is administered to a subject, and the genetically modified HSPC comprises (i) an extracellular component containing a ligand binding domain that binds a selectively expressed cell marker, and (ii) an intracellular component containing an effector domain. And targeting at least one cell marker that is selectively expressed on cancer cells from the subject of interest.
93. Further comprising administering to the subject a therapeutically effective amount of HSPC to treat the subject's immunodeficiency, pancytopenia, neutropenia, and / or leukopenia. The method according to any one of forms 90-92.
94. Embodiment 93. The immunodeficiency, pancytopenia, neutropenia, and / or leukopenia are due to chemotherapy, radiation therapy, and / or bone marrow abolition therapy for HCT. Method.
95. The method according to any one of embodiments 90-94, wherein said cell marker is CD19, ROR1, PSMA, PSCA, mesothelin, CD20, WT1, or Her2.
96. The method of any one of embodiments 90-95, wherein prior to administration no immunological adaptation to the subject is required.
97. The method of any one of embodiments 90-96, wherein said unwanted cancer cells are CD19-expressing acute lymphocytic leukemia cells.
98. The method of any one of embodiments 90-97, wherein said subject is a patient with recurrent childhood acute lymphoblastic leukemia.
99. Immunization of a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of HSPC and / or genetically modified HSPC, thereby increasing the immune system of the subject. How to grow the system.
100. The method of embodiment 99, wherein said proliferation is required based on one or more of immunodeficiency, pancytopenia, neutropenia, or leukopenia.
101. 101. The method of embodiment 99 or 100, wherein said growth is required based on one or more of viral infection, microbial infection, parasitic infection, renal disease, and / or renal failure.
102. The method of any one of embodiments 99-101, wherein said proliferation is required based on chemotherapy, HCT-enabled bone marrow abolition therapy, and / or exposure to acute ionizing radiation.
103. 103. The method of any one of embodiments 99-102, wherein said proliferation is required based on exposure to an agent that causes myelosuppression or hematopoietic defects.
104. Any one of embodiments 99-103 wherein said proliferation is required based on exposure to penicillin, gancyclovir, daunomycin, meprobamate, aminopyrine, dipyrone, phenytoin, carbamazepine, propylthiouracil, and / or methimazole. The method described.
105. The method of any one of embodiments 99-104, wherein said proliferation is required based on exposure to dialysis.
106. Any of Embodiments 99-105 further comprising targeting unwanted cancer cells in the subject by administering genetically modified HSPC and / or genetically modified non-effector T cells. The method of claim 1, wherein the genetically modified HSPC and / or the genetically modified non-effector T cell is (i) selectively expressed on a cancer cell of the subject. Expressing an extracellular component comprising a ligand binding domain that binds a known cellular marker and (ii) an intracellular component comprising an effector domain.
107. The cancer cells are adrenal gland cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, carcinoma, cervical cancer, colon cancer, colon cancer, endometrial cancer, Ear, nose and throat (ENT) cancer, endometrial cancer, esophageal cancer, digestive system cancer, head and neck cancer, Hodgkin's disease, intestinal cancer, kidney cancer, laryngeal cancer, leukemia, liver Cancer, lymph node cancer, malignant lymphoma, lung cancer, melanoma, mesothelioma, myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, penis Cancer, pharyngeal cancer, prostate cancer, rectal cancer, sarcoma, seminoma, skin cancer, gastric cancer, teratoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, vascular tumor, and / or them 107. The method of embodiment 106, wherein the cells are from metastases of.
108. The cell markers are A33, BAGE, Bcl-2, β-catenin, B7H4, BTLA, CA125, CA19-9, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD44v6, CD45, CD123, CEA, CEACAM6. , C-MET, CS-1, cyclin B1, DAGE, EBNA, EGFR, ephrin B2, ErbB2, ErbB3, ErbB4, EphA2, estrogen receptor, FAP, ferritin, α-fetoprotein (AFP), FLT1, FLT4, folate binding. Protein, Frizzled, GAGE, G250, GD-2, GHRHR, GHR, GM2, gp75, gp100 (Pmel 17), gp130, HLA, HER-2 / neu, HPV E6, HPV E7, human terrorism. Reverse transcriptase, HVEM, IGF1R, IL6R, KDR, Ki-67, LIFRβ, LRP, LRP5, LTβR, mesothelin, OSMRβ, p53, PD1, PD-L1, PD-L2, PRAME, progesterone receptor, PSA, PSMA , PTCH1, MAGE, MART, Mesothelin, MUC, MUC1, MUM-1-B, myc, NYESO-1, RANK, ras, Robo1, RORl, survivin, TCRα, TCRβ, tenascin, TGFBR1, TGFBR2, TLR7, TLR9, TLR9, TLR9. 108. The method of embodiment 106 or 107, which is a cell marker selected from, TNFR2, TNFRSF4, TWEAK-R, TSTA tyrosinase, VEGF, and WT1.
109. The cancer is leukemia / lymphoma and the cellular marker is one or more of CD19, CD20, CD22, ROR1, CD33, and WT-1, the cancer is multiple myeloma, and The marker for the cells is BCMA, the cancer is prostate cancer and the cell marker is one or more of PSMA, WT1, PSCA, and SV40 T, the cancer is breast cancer, and The cell marker is one or more of HER2, ERBB2, and ROR1, the cancer is stem cell cancer and the cell marker is CD133, the cancer is ovarian cancer, and the cell marker is , L1-CAM, MUC-CD, folate receptor, Lewis Y, ROR1, mesothelin, and WT-1 are one or more, and the cancer is mesothelium. Yes and the cell marker is mesothelin, the cancer is renal cell cancer and the cell marker is CAIX, the cancer is melanoma and the cell marker is GD2, The cancer is pancreatic cancer and the cell marker is one or more of mesothelin, CEA, CD24, and ROR1, or the cancer is lung cancer and the cell marker is ROR1. The method according to any one of aspects 106-108.
110. The method according to any one of embodiments 106-109, wherein said cancer is acute lymphocytic leukemia and said subject is a pediatric patient.
111. The method according to any one of embodiments 106-110, wherein no immunological adaptation to the subject is required prior to administration.
112. A therapeutically effective amount of the genetically modified HSPC and / or the genetically modified non-effector T cells is administered to the subject as needed, and the genetically modified cells are ( Destruction, comprising targeting and destroying cells expressing i) an extracellular component containing the ligand binding domain CD19 and (ii) an intracellular component containing an effector domain, thereby selectively expressing CD19. Targeting cells that selectively express CD19 in response to.
113. Embodiments further comprising the treatment of immunodeficiency, pancytopenia, neutropenia, and / or leukopenia in the subject by administering to the subject a therapeutically effective amount of HSPC. The method according to 112.
114. Embodiment 113. The immunodeficiency, pancytopenia, neutropenia, and / or leukopenia are due to chemotherapy, radiation therapy, and / or bone marrow abolition therapy for HCT. the method of.
115. The method of any one of embodiments 112-114, wherein immunological adaptation to the subject is not required prior to administration.
116. The method of any one of embodiments 112-115, wherein the cells selectively expressing CD19 are acute lymphocytic leukemia cells.
117. The method of any one of embodiments 112-116, wherein said subject is a recurrent childhood acute lymphoblastic leukemia patient.

  Example 1. We created a 2/3 generation lentiviral vector design and cGMP production that corresponded to the synchronized expression of CD19-CAR and huEGFRt selection / self-destructing constructs. Both the SIN vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vector under cGMP conditions encoding a CD19-specific CAR and huEGFRt, a truncated human EGFR lacking the intracellular signaling domain, Was expressed. The CD19-specific scFvFc-CD3ζCD28 CAR and huEGFRt vectors consist of a hybrid 5′LTR in which the U3 region is replaced by the CMV promoter and a 3′LTR in which the cis-acting control sequence is completely removed from the U3 region. including. As a result, both the 5'and 3'LTRs are inactivated when the provirus is produced and integrated into the chromosome. The CD19 CAR is a human IgG1 mAb (FMC63) specific for CD19, hinge region of Fc and human IgG4 heavy chain, human CD28 transmembrane region, and human GMCSFR α chain leader sequence derived from cytoplasmic domains of CD3ζ and CD28, VL and VH. Contains an array. This construct was cloned into modified pHIV7 in the region where its CMV promoter was replaced with the human EF-1α promoter (FIG. 29A). The vector allows for approximately 1: 1 expression of CD19 CAR and huEGFRt through the use of the T2A element. Next, a CD19-specific scFv-4-1BB / CD3ζ CAR fragment was derived from an IgG1-mouse monoclonal antibody (FMC63), a human IgG4 hinge and a human CD28 transmembrane region, and a 4-1BB co-stimulatory element with a cytoplasmic tail of human CD3ζ. It encodes the N-terminal leader peptide of the human GMCSF receptor alpha chain signaling sequence to direct induced surface expression of CD19-specific scFv (Figure 29B). Again, the vector allows for approximately 1: 1 expression of CD19 CAR and huEGFRt through the use of the T2A element.

  Expression of huEGFRt is given to a second cell surface marker that allows easy experiments of transduction efficiency. Biotinylated arbitux binds to huEGFRt expressed on the cell surface and can be labeled with a fluorescent dye for analysis by flow cytometry. In addition, it can be used as a self-destructing gene in clinical settings involving the treatment of Erbitux. At the CAR location, a similar vector with eGFP was produced.

  Example 2. Notch-mediated in vitro proliferation of CB HSPC is a clinically validated, benign cell therapy product that can provide its benefits at any time without HLA matching, and of HCT and intensive chemotherapy. In both situations, provide temporary bone marrow engraftment. Off-the-shelf growth units were infused in over 85 subjects, but no serious adverse events were noted, except for one case of DMSO-induced allergic reaction. Furthermore, there was no permanent engraftment over 180 days in the HCT scene and 14 days after infusion in the chemotherapy scene.

Method. A cord blood / placental blood unit was collected from humans at birth. The collected blood was mixed and stored with an anticoagulant to prevent clotting. Prior to the start of planned growth cultures, tissue culture vessels were placed in phosphate buffered saline (PBS) at 2.5 μg / ml Delta1 ^ext- lgG and 5 μg / ml RetroNectin® ( The first coating was carried out with recombinant human fibronectin fragment) (Clontech Laboratories, Inc., Madison, Wis.) Overnight at 4 ° C. or at 37 ° C. for a minimum of 2 hours. The flask was then washed with PBS and then blocked with PBS-2% human serum albumin (HSA). Separation of red blood cells from a fresh cord blood unit and sorting of CD34 ⁺ cells using an automated MACS® Cell Separation System (Miltenyi Biotec GmbH, Gladbach, Germany). Was carried out. After enrichment, the percentage of CD34 ⁺ cells in the sample was increased relative to the percentage of CD34 ⁺ cells in the sample before enrichment. Fragments of the enriched CD34 ⁺ cells were isolated from rhIL-3 (10 ng / ml), rhIL-6 (50 ng / ml), rhTPO (50 ng / ml), rhFlt-3L (50 ng / ml), rhSCF (50). ng / ml) in supplemented with StemSpan ^TM serum-free growth medium ^{(STEMSPAN TM serum free Expansion medium)} (StemCell Technologies, Vancouver, consisting British Columbia), and resuspended in the final medium.

  The SIN lentiviral vector directing co-growth of the CD19-specific scFvFc: CD28: ζ chimeric antigen receptor and the selective self-destructing construct huEGFRt was treated with lentiviral supernatant (MOI 3) and 4 on day 3 or 4 thereof. Notch-grown CB stem cells were transduced with 800 × g together with μg / ml protamine sulfate via centrifugation at 32 ° C. for 45 minutes. Also, the SIN lentiviral vector encoded 4-1BB costimulation (see overview in the figure). Considering the expression of CAR on HSPC with potential signaling capacity, irradiated LCL was added to the cultures on day 7 at a 1: 1 ratio to give antigenic stimulation.

  At the final stage of the expansion culture, NK cells and neutrophils are still immature. The culture method was devised to enhance maturity for a complete evaluation of the lytic capacity. For NK cells, the culture was supplemented with human serum, RPMI medium supplemented with 50 U / mL IL-2 and 500 ng / mL IL-15, or human serum, L-glutamine, 50 U / mL IL. -2 and RPMI medium supplemented with 500 ng / mL IL-15.

The NOD / SCID IL2R null (NOG) mouse model was used to assess engraftment of proliferating CB cells. After sublethal irradiation, mice can be reliably engrafted with expanded CB cells. To determine engraftment in transduced expanded CB cells, NOG mice were irradiated with a linear accelerator at a dose of 325 cGy and cultured from 10,000 to 30,000 cells on Delta-1 ^ext-IgG. The products produced from a number of CD34 ⁺ CB cells were infused via tail vein injection.

  result. Transduction efficiencies ranged from 10 to> 50% and between CD34 + and CD34-, there was generally equal transduction. This was consistent with the FDA requirements for clinical gene therapy cell products, as copy number analysis showed between 1-4 copies / cell as determined by validated quantitative real-time PCR analysis. There is.

  CD34 + CB cells cultured on Notch ligand contain a variety of cell types, which can be identified based on immunophenotyping. Cultures transduced with the CD19 CAR lentivirus were compared to non-transduced cultures from the same cord blood unit, but overall growth of cells at the time of harvest or in cultures containing CD34-fold and TNC-fold growth. No significant difference was detected by the final immunophenotyping in.

  Expression of the transgene did not affect the phenotype of the final culture on day 14, and expression of the transgene was found in a subset of all cells and appeared relatively stable over the culture period. .

  To determine if exposure to the antibody caused adverse effects on the culture, additional experiments were performed in which the cell culture was exposed to CD19 + LCL. On day 7, there was no inconvenience due to the addition of irradiated LCL to the cultures in a 1: 1 ratio, and in fact in both transduced and non-transduced cultures, growth and viability Was raised. The LCL does not show an increase in CAR + number, suggesting that the antigen does not enhance the proliferation of CAR expressing immature cells. Furthermore, the transgene was detected equally in all plasma cell subsets of the final product. See Figures 30A, 30B, 31, 32 and 33 for graphical depiction of these results.

  Introduction of effector functions by encountering CD19 via expression of the CD19 CAR is important for the ultimate anti-cancer activity (eg, anti-leukemia) of the modified CB HSPC cells. Differentiated culture conditions resulted in an increase in NK cells (Figure 34). Fragments of CD56 + cells were sorted and used for CRA with K562 and LCL target cells. As expected, both untransduced and transduced cells were able to kill K562, and LCL was also killed by both, but through the expression of the CAR, LCL lysis was greatly enhanced. It was More specifically, the CD19-CAR expressing NK cells had enhanced cytotoxic activity compared to non-transduced NK cells, whereas both equally killed the K562 target (75% vs. 80%). (50% vs. 30%). See FIG. 35.

  The NOG model transplanted with proliferating CB cells resulted in the proliferation of large numbers of CD19 + cells beginning about 4-5 weeks after transplantation. There was no effect on the early engraftment of transduced cells, but there was a significant reduction in CD19 engraftment in mice transplanted with cells expressing CD19 CAR compared to non-transduced cells. , The number of CD19 cells was> 20% of the engrafted cells showing anti-CD19 activity. The number of NK cells was increased by the use of NSO-IL15 secreting cells by irradiation and subcutaneous injection from the 3rd week of initiation to 3rd week to give high effector function. This effect increased the amount of CD56 + cells in vivo. See Figures 36 and 37.

This data shows that transduction of proliferating CB cells in culture in the presence of immobilized Delta ^1ext-IgG to express a CD19-specific CAR ^resulted in their proliferation or in their regenerative capacity in the mouse model. Also show no qualitative and quantitative detectable effects. These results indicate that genetically engineered graft-versus-cancer effects (eg leukemia) into cord blood transplants hold promise. Furthermore, transduction of CD19 CAR into CB HSPC-expanding universal donors is immediate-acting, according to the identification of subjects with a clinical need for treatment, such as those with relapse or permanent MRD. Infusion of anti-CD19 product is possible to provide (eg, pre-dose immunological compatibility is not required). CD34 + umbilical cord blood cells grown on Notch ligand, with no effect on whole culture or in vivo engraftment capacity, showed reliable transduction while simultaneously genetically incorporating anti-CD19 activity. Since expanded cord blood cells have already been used clinically as an existing, non-HLA-matched cell therapy, the examples described herein provide immunotherapy for patients who are inaccessible. It shows the use as a further ready-to-use cell therapy, which makes it possible to receive and genetically obtain autologous T cell products.

  As indicated, the practice of the present disclosure may incorporate conventional methods of virology, microbiology, molecular biology, and recombinant DNA technology in the art, unless otherwise indicated. Such techniques are explained fully in the literature. For example, Sambrook, et al. Molecular Cloning: A Laboratory Manual (Current Edition), DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.), Oligonucleotide Synthesis (N. Gait, ed., Current Edition), Nucleic Acid Hybridization (B. Hamestritions, C., Ed.). Hames & S. Higgins, eds., Current Edition), CRC Handbook of Parvoviruses, vol. I & II (P. Tijesen, ed.), Fundamental Virology, 2nd Edition, vol. See I & II (BN Fields and DM Knipe, eds.). The teachings for each and the same purpose, each set forth, are hereby incorporated by reference.

  As will be appreciated by one of skill in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of, the elements, steps, inclusions or compositions particularly shown therein. You can “Includes” or “including” means “comprises, consisting essentially of or consisting of”. The transition terms “comprise” or “comprises” are meant to include, but are not limited to, and in non-limiting manner, and even in large amounts, non-specific elements, steps, inclusions, or compositions. Allows inclusion of things. The transition phrase "consisting of" excludes any element, step, inclusion, or composition not specified. The transition phrase "consisting essentially of" limits the scope of an embodiment to a particular element, step, inclusion, or composition, and to those that do not materially affect the embodiment. To do. The material effect is (i) a statistically significant reduction in the effectiveness of cell administration to create an anti-cancer effect in the subject, and / or (ii) to proliferate the subject's immune system. It may result in a statistically significant reduction in the effectiveness of cell administration.

  Unless otherwise indicated, all numbers indicating characteristics such as amount of inclusions, molecular weight, reaction conditions, and used in the specification and claims hereinafter are modified in all examples by the term "about". Is understood. Accordingly, unless expressly stated to the contrary, the numerical parameters recited in the specification and appended claims are approximations that may vary depending on the desired features that are considered to be obtained by the present invention. At least each numerical parameter should be construed in light of the number of significant digits reported and by application of conventional rounding techniques, not as an attempt to limit the application of the doctrine of equivalents to the scope of the claim. . Where further clarification is required, the term "about", when used in combination with a stated numerical value or range of numerical values, has the meaning that one of ordinary skill in the art reasonably considers to belong, i.e., stated. Within ± 20% of the asserted value, ± 19% of the asserted value, ± 18% of the asserted value, ± 17% of the asserted value compared to the specified numerical value or range of numerical values. Within ± 16% of the asserted value, Within ± 15% of the asserted value, Within ± 14% of the asserted value, Within ± 13% of the asserted value Within ± 12% of the stated value, within ± 11% of the stated value, within ± 10% of the stated value, within ± 9% of the stated value, within ± 8% of the stated value Within ± 7% of the asserted value, Within ± 6% of the asserted value, Within ± 5% of the asserted value, Within ± 4% of the asserted value ± % Within the range, at within ± 2% of assertion values or within a range of ± 1% of the stated value, indicates that somewhat more or less.

  Despite the broad numerical ranges and parameters of the present invention which are approximations, the numerical values set forth in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective test measurements.

  The terms “a”, “an”, “the” and like directives, as used in the context of describing the invention, especially in the context of the claims that follow, are: It is intended that the specification includes both the singular and the plural unless specifically stated otherwise or dictated by the context. The recitation of range of values herein is intended only to serve as a shorthand notation for each individual value contained within that range. Unless otherwise specified herein, individual values are incorporated herein as if they were individually described herein. All methods described herein can be carried out in any suitable order, unless explicitly stated otherwise herein or unless the context clearly dictates otherwise. Any and all exemplifications or uses of the exemplary terms (eg, “such as”) provided herein are intended only to better illuminate the invention and other than claimed invention. It does not limit the scope. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

  Groups of alternative elements or embodiments of the invention disclosed herein are not to be construed as constraints. Each group member may refer to and separately claim or be in any combination with another member or other element of the group found herein. It is anticipated that one or more personnel of a group may be included in or deleted from the group for convenience and / or patentability reasons. In the event that any such inclusion or deletion occurs, the specification is considered to include that group, as amended to meet the written description of all Markush groups used in the appended claims. .

  Particular embodiments of the invention have been described, including the best mode known to the inventors for practicing the invention. Of course, modifications of the contents described in the embodiments will be apparent to those skilled in the art reading the above description. The inventor expects that those skilled in the art will appropriately incorporate such modifications, and that the inventor intends that the present invention be practiced other than as specifically described herein. is doing. Accordingly, this invention includes all modifications and equivalents of the subject matter repeated in the claims appended hereto within the scope of applicable law. Moreover, any combinations of the elements described above in all possible variations are included in the invention, unless explicitly stated otherwise herein or unless the context clearly dictates.

  Furthermore, numerous references have been obtained throughout the specification to books, magazine articles, papers, patents, printed publications, and the like (to be precise, the "reference section"). Each of the above references is individually incorporated by reference herein for its teachings.

  Finally, it will be appreciated that the embodiments of the invention described herein are illustrative of the principles of the invention. Other changes that may be incorporated are within the scope of the invention. Thus, by way of example, and not limitation, other configurations of the present invention may be utilized in accordance with the teachings herein. Therefore, the present invention is not limited to that precisely as shown and described.

  The details presented herein are by way of example and for the purposes of exemplary discussion of preferred embodiments of the invention only, and are most useful and the principles of the invention and concepts of various embodiments. It is presented to provide what is considered an easily understandable description of the physical aspect. In this regard, there is no attempt to present details of the construction of the invention in the more detail that is necessary for a basic understanding of the invention, and one of ordinary skill in the art will appreciate what forms of the invention are. The description incorporated in the drawings and / or illustrations that clarify such may be embodied in practice.

  The definitions and explanations used in this disclosure modify the following examples in a clear and unambiguous manner or the application of their meaning makes any configuration meaningless or essentially meaningless. Unless and unless the case, it is meant and intended to constrain any future configuration. Where the construction of the term renders the term meaningless or essentially meaningless, the definition is Webster's Dictionary, 3rd Edition, or the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Should be taken from dictionaries known to those skilled in the art, such as University Press, Oxford, 2004).

Claims (30)

(1) A therapeutically effective amount of a genetically unmodified hematopoietic stem progenitor cell (HSPC),
(2) (a) a therapeutically effective amount of a genetically modified HSPC and / or (b) a therapeutically effective amount of a genetically modified myeloid stem / progenitor cell and gene A formulation comprising a genetically modified lymphocyte stem / progenitor cell comprising:
The genetically modified cells in (2) are not immunologically compatible with the subject and are (i) to (iii) below,
(I) an extracellular component comprising a ligand binding domain that binds a cell marker selectively expressed on undesired cells, the ligand binding domain being CD19, ROR1, PSMA, PSCA, mesothelin, CD20, WT1, Her2. Or bound to CD123,
(Ii) a transmembrane domain linked to an extracellular component, and
(Iii) an intracellular component linked to a transmembrane domain, the intracellular component comprising an effector domain containing an intracellular signal transduction domain of CD3ζ, CD28 or 4-1BB,
A formulation expressing a molecule comprising:   The formulation of claim 1, wherein the ligand binding domain is an antibody fragment.   The formulation of claim 1, wherein the ligand binding domain is a single chain variable fragment of an antibody.   The ligand-binding domain is the sequence of RASQDISKYLN (SEQ ID NO. 108), CDRL1, SRLHSGV (SEQ ID NO. 111), CDRL2, and GNTLPYTFG (SEQ ID NO. 104) according to Kabat numbering. CDRL3, DYGVS (SEQ ID NO. 103) sequence CDRH1, VIWGSETTYNYNSALKS (SEQ ID NO. 114) sequence CDRH2, and YAMDYWG (SEQ ID NO. 115) sequence CDRH3, single-chain Fv. The formulation of claim 1, which is a fragment (scFv).   The ligand binding domain is SEQ ID NO. The formulation of claim 1, which is a single chain Fv fragment (scFv) comprising the CDRs of the light and heavy chain variable regions of the FMC63 antibody represented by 108, 111, 104, 103, 114 and 115.   The ligand binding domain is SEQ ID NO. The formulation of claim 1, which is an scFv comprising the CDRs of the heavy and light chain variable regions of the single chain Fv fragment (scFv) of R12 represented by 56, 57 or 58.   The ligand binding domain is SEQ ID NO. The formulation of claim 1, wherein the formulation is scFv comprising the CDRs of the heavy and light chain variable regions of the single chain Fv fragment (scFv) of R11 represented by 53, 54 or 55. Effector domains are C ARD11, CD3γ, CD3δ, CD3ε , C D27 , C D79A, CD79B, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTPT, and PPTα. , OX40, ROR2, Ryk, SLAMF1 , Slp76, further comprising TCR a, TCR [beta], TRIM, 1 or more selected from Wnt and Zap70 signaling and / or stimulating domain, the signaling and / or stimulating domain,請 Motomeko 1 The formulation according to. Effector domain, CD27, O X40, CD30, CD40, lymphocytes functional - associated antigen -1 (LFA-1), CD2 , CD7, LIGHT, one or more co-stimulatory domain selected from NKG2C and B7-H3 further The formulation of claim 1, comprising: Intracellular components
(I) all or part of the CD3ζ signaling domain,
(Ii) all or part of the signaling domain of CD28,
(Iii) an effector comprising an intracellular signaling domain comprising all or part of the 4-1BB signaling domain, or (iv) CD3ζ, CD28 and / or all or part of the 4-1BB signaling domain. Including the domain,
The formulation of claim 1.   The formulation of claim 1, wherein the intracellular component comprises an effector domain comprising a variant of CD3ζ and / or part of the intracellular signaling domain of 4-1BB.   The formulation of claim 1, wherein the molecule further comprises a spacer region.   13. The formulation of claim 12, wherein the spacer region comprises part of the hinge region of a human antibody.   13. The formulation of claim 12, wherein the spacer region comprises a hinge region and at least one other portion of the Fc domain of a human antibody selected from CH1, CH2, CH3 or combinations thereof.   13. The formulation of claim 12, wherein the spacer region comprises an Fc domain and a human IgG4 heavy chain hinge.   The formulation according to claim 12, wherein the spacer region is a region having a length selected from 12 or less amino acids, 119 or less amino acids, or 229 or less amino acids.   13. The formulation of claim 12, wherein the spacer region has the sequence represented by SEQ ID NO: 47, SEQ ID NO: 52, or SEQ ID NO: 61. The formulation of claim 1 , wherein the transmembrane domain is the CD28 transmembrane domain or the CD4 transmembrane domain.   The formulation of claim 1, wherein the extracellular component further comprises a tag sequence. The extracellular component comprises a ligand binding domain that binds CD19 and the intracellular component comprises an effector domain that comprises the cytoplasmic domain of CD28 or 4-1BB,
The formulation of claim 1, wherein the molecule further comprises a spacer region comprising the hinge region of human IgG4, and a human CD4 or CD28 transmembrane domain.   The genetically modified cell expresses a chimeric antigen receptor (CAR) having a sequence represented by SEQ ID NO: 34, 53, 54, 55, 56, 57, or 58. Formulation.   The formulation according to claim 1, which is formulated for infusion or injection. (1) A therapeutically effective amount of a genetically unmodified HSPC,
(2) (a) a therapeutically effective amount of a genetically modified HSPC and / or (b) a therapeutically effective amount of a genetically modified myeloid stem / progenitor cell and gene A medicinal product comprising a genetically modified lymphocyte stem / progenitor cell comprising:
The genetically modified cells in (2) are not immunologically compatible with the subject and are (i) to (iii) below,
(I) an extracellular component comprising a ligand binding domain that binds a cell marker selectively expressed on undesired cells, the ligand binding domain being CD19, ROR1, PSMA, PSCA, mesothelin, CD20, WT1, Her2. Or bound to CD123,
(Ii) a transmembrane domain linked to an extracellular component and
(Iii) an intracellular component linked to a transmembrane domain, the intracellular component comprising an effector domain containing an intracellular signal transduction domain of CD3ζ, CD28 or 4-1BB,
A medicament which expresses a molecule comprising, whereby the immune system of a subject repopulates and targets unwanted cells. 24. The medicament of claim 23 , which requires no immunocompatibility in the subject prior to administration. 25. The medicament according to claim 24 , wherein the cells are non-autologous to the subject. The medicine according to claim 23 , wherein the subject is a patient with recurrent childhood acute lymphocytic leukemia. Repopulation of the immune system treats immunodeficiency, pancytopenia, neutropenia, and / or leukopenia by chemotherapy, radiation therapy, and / or bone marrow abolition therapy in response to HCT, The medicine according to claim 23 . 24. The medicament of claim 23 , wherein repopulation is required based on exposure to agents that cause myelosuppression or hematopoietic defects. The need for repopulation to be based on exposure to bone marrow abolition therapy in response to hematopoietic cell transplantation (HCT) and the unwanted cells are acute lymphocytic leukemia cells expressing CD19. Item 23. The medicament according to Item 23 . The medicine according to claim 23 , wherein the unwanted cells are unwanted cancer cells.

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